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1.
Arch Biochem Biophys ; 737: 109534, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36740034

RESUMO

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from -90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current. In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes. After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications. Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.


Assuntos
Bloqueadores dos Canais de Potássio , Potássio , Humanos , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Cloretos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Pulmão/metabolismo , Estresse Oxidativo
2.
Cancers (Basel) ; 15(1)2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36612174

RESUMO

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

3.
Toxicol Lett ; 338: 40-50, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33279629

RESUMO

Air pollution represents one of the main risks for both environment and human health. The rapid urbanization has been leading to a continuous release of harmful manmade substances into the atmosphere which are associated to the exacerbation of several pathologies. The skin is the main barrier of our body against the external environment and it is the main target for the outdoor stressors. Among the pollutants, Ozone (O3) is one of the most toxic, able to initiate oxidative reactions and activate inflammatory response, leading to the onset of several skin conditions. Moreover, skin is daily subjected to the activity of Ultraviolet Radiation which are well known to induce harmful cutaneous effects including skin aging and sunburn. Even though both UV and O3 are able to affect the skin homeostasis, very few studies have investigated their possible additive effect. Therefore, in this study we evaluated the effect of the combined exposure of O3 and UV in inducing skin damage, by exposing human skin explants to UV alone or in combination with O3 for 4-days. Markers related to inflammation, redox homeostasis and tissue structure were analyzed. Our results demonstrated that O3 is able to amplify the UV induced skin oxinflammation markers.


Assuntos
Ozônio/toxicidade , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proteínas Filagrinas , Humanos , Mediadores da Inflamação/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Pele/metabolismo , Pele/patologia , Proteínas de Junções Íntimas/metabolismo , Técnicas de Cultura de Tecidos
4.
J Cell Physiol ; 234(10): 17704-17713, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30805940

RESUMO

K+ channels of the alveolar epithelium control the driving force acting on the ionic and solvent flow through the cell membrane contributing to the maintenance of cell volume and the constitution of epithelial lining fluid. In the present work, we analyze the effect of the Cl- channel inhibitors: (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-inden-5-yl)oxy] butanoic acid (DCPIB) and 9-anthracenecarboxylic acid (9-AC) on the total current in a type II pneumocytes (A549 cell line) model by patch clamp, immunocytochemical, and gene knockdown techniques. We noted that DCPIB and 9-AC promote the activation of K conductance. In fact, they significantly increase the intensity of the current and shift its reversal potential to values more negative than the control. By silencing outward rectifier channel in its anoctamin 6 portion, we excluded a direct involvement of Cl- ions in modulation of IK and, by means of functional tests with its specific inhibitor spadin, we identified the TREK-1 channel as the presumable target of both drugs. As the activity of TREK-1 has a key role for the correct functioning of the alveolar epithelium, the identification of DCPIB and 9-AC molecules as its activators suggests their possible use to build new pharmacological tools for the modulation of this channel.


Assuntos
Células Epiteliais Alveolares/metabolismo , Cloretos/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Células A549 , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Tamanho Celular , Canais de Cloreto/metabolismo , Humanos , Técnicas de Patch-Clamp/métodos
5.
Neuroscience ; 385: 198-214, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29913242

RESUMO

The role of glutamate in quantal release at the cytoneural junction was examined by measuring mEPSPs and afferent spikes at the posterior canal in the intact frog labyrinth. Release was enhanced by exogenous glutamate, or dl-TBOA, a blocker of glutamate reuptake. Conversely, drugs acting on ionotropic glutamate receptors did not affect release; the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) blocker CNQX decreased mEPSP size in a dose-dependent manner; the NMDA-R blocker d-AP5 at concentrations <200 µM did not affect mEPSP size, either in the presence or absence of Mg and glycine. In isolated hair cells, glutamate did not modify Ca currents. Instead, it systematically reduced the compound delayed potassium current, IKD, whereas the metabotropic glutamate receptor (mGluR)-II inverse agonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495), increased it. Given mGluR-II decrease cAMP production, these finding are consistent with the reported sensitivity of IKD to protein kinase A (PKA)-mediated phosphorylation. LY341495 also enhanced transmitter release, presumably through phosphorylation-mediated facilitation of the release machinery. The observed enhancement of release by glutamate confirms previous literature data, and can be attributed to activation of mGluR-I that promotes Ca release from intracellular stores. Glutamate-induced reduction in the repolarizing IKD may contribute to facilitation of release. Overall, glutamate exerts both a positive feedback action on mGluR-I, through activation of the phospholipase C (PLC)/IP3 path, and the negative feedback, by interfering with substrate phosphorylation through Gi/0-coupled mGluRs-II/III. The positive feedback prevails, which may explain the increase in overall rates of release observed during mechanical stimulation (symmetrical in the excitatory and inhibitory directions). The negative feedback may protect the junction from over-activation.


Assuntos
Orelha Interna/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Células Ciliadas Auditivas/efeitos dos fármacos , Sinapses/efeitos dos fármacos , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Aminoácidos/farmacologia , Animais , Anuros , Ácido Aspártico/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas de Patch-Clamp , Receptores Ionotrópicos de Glutamato/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Xantenos/farmacologia
6.
J Cell Physiol ; 233(8): 6018-6027, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29271475

RESUMO

The lung tissue is one of the main targets of oxidative stress due to external sources and respiratory activity. In our previous work, we have demonstrated in that O3 exposure alters the Cl- current-voltage relationship, with the appearance of a large outward rectifier component mainly sustained by outward rectifier chloride channels (ORCCs) in human lung epithelial cells (A549 line). In the present study, we have performed patch clamp experiments, in order to identify which one of the O3 byproducts (4hydroxynonenal (HNE) and/or H2 O2 ) was responsible for chloride current change. While 4HNE exposition (up to 25 µM for 30' before electrophysiological analysis) did not reproduce O3 effect, H2 O2 produced by glucose oxidase 10 mU for 24 hr before electrophysiological analysis mimicked O3 response. This result was confirmed treating the cell with catalase (CAT) before O3 exposure (1,000 U/ml for 2 hr): CAT was able to rescue Cl- current alteration. Since CAT is regulated by Nrf2 transcription factor, we pre-treated the cells with the Nrf2 activators, resveratrol and tBHQ. Immunochemical and immunocytochemical results showed Nrf2 activation with both substances that lead to prevent OS effect on Cl- current. These data bring new insights into the mechanisms involved in OS-induced lung tissue damage, pointing out the role of H2 O2 in chloride current alteration and the ability of Nfr2 activation in preventing this effect.


Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Pulmão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Células A549 , Antioxidantes/metabolismo , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Pulmão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ozônio/farmacologia
7.
Neuroscience ; 357: 20-36, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28576732

RESUMO

The post-transductional elaboration of sensory input at the frog semicircular canal has been studied by correlating the effects of drugs that interfere with phosphorylation processes on: (i) potassium conductances in isolated hair cell and (ii) transmitter release at the cytoneural junction in the intact labyrinth. At hair cells, delayed potassium currents (IKD) undergo voltage- and time-dependent inactivation; inactivation removal requires ATP, is sensitive to kinase blockade, but is unaffected by exogenous application of cyclic nucleotides. We report here that forskolin, an activator of endogenous adenylyl cyclase, enhances IKD inactivation removal in isolated hair cells, but produces an overall decrease in IKD amplitude consistent with the direct blocking action of the drug on several families of K channels. In the intact labyrinth, forskolin enhances transmitter release, consistent with such depression of K conductances. Kinase blockers - H-89 and KT5823 - have been shown to reduce IKD inactivation removal and IKD amplitude at isolated hair cells. In the labyrinth, the effects of these drugs on junctional activity are quite variable, with predominant inhibition of transmitter release, rather than the enhancement expected from the impairment of K currents. The overall action of forskolin and kinase inhibitors on K conductances is similar (depression), but they have opposite effects on transmitter release: this indicates that some intermediate steps between the bioelectric control of hair cell membrane potential and transmitter release are affected in opposite ways and therefore are presumably regulated by protein phosphorylation.


Assuntos
Carbazóis/farmacologia , Colforsina/farmacologia , Células Ciliadas da Ampola/efeitos dos fármacos , Isoquinolinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Proteínas de Anfíbios/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Células Ciliadas da Ampola/metabolismo , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/metabolismo , Rana esculenta , Técnicas de Cultura de Tecidos
8.
J Comput Neurosci ; 42(2): 123-131, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27844245

RESUMO

A computational model has been developed to simulate the electrical behavior of the type II hair cell dissected from the crista ampullaris of frog semicircular canals. In its basolateral membrane, it hosts a system of four voltage-dependent conductances (g A , g KV , g KCa , g Ca ). The conductance behavior was mathematically described using original patch-clamp experimental data. The transient K current, IA, was isolated as the difference between the currents obtained before and after removing IA inactivation. The remaining current, IKD, results from the summation of a voltage-dependent K current, IKV, a voltage-calcium-dependent K current, IKCa, and the calcium current, ICa. IKD was modeled as a single lumped current, since the physiological role of each component is actually not discernible. To gain a clear understanding of its prominent role in sustaining transmitter release at the cytoneural junction, ICa was modeled under different experimental conditions. The model includes the description of voltage- and time-dependent kinetics for each single current. After imposing any starting holding potential, the system sets the pertinent values of the variables and continually updates them in response to variations in membrane potential. The model reconstructs the individual I-V curves obtained in voltage-clamp experiments and simulations compare favorably with the experimental data. The model proves useful in describing the early steps of signal processing that results from the interaction of the apical receptor current with the basolateral voltage-dependent conductances. The program is thus helpful in understanding aspects of sensory transduction that are hard to analyze in the native hair cell of the crista ampullaris.


Assuntos
Células Ciliadas Auditivas , Modelos Neurológicos , Canais Semicirculares , Cálcio , Cabelo , Potenciais da Membrana , Técnicas de Patch-Clamp , Transdução de Sinais
9.
J Cell Physiol ; 232(7): 1817-1825, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27886375

RESUMO

Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution, and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre-treatment. Whole-cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I-V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 h. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT-qPCR analysis showed an increase in ClC-2 and ORCC mRNA after ozone exposure. In addition, pre-treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation. Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above-mentioned damage. J. Cell. Physiol. 232: 1817-1825, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Pulmão/patologia , Estresse Oxidativo , Células A549 , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Ozônio/farmacologia , Técnicas de Patch-Clamp , Substâncias Protetoras/farmacologia , Vitamina E/farmacologia
10.
Front Cell Neurosci ; 9: 235, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26157360

RESUMO

At the frog semicircular canals, the afferent fibers display high spontaneous activity (mEPSPs), due to transmitter release from hair cells. mEPSP and spike frequencies are modulated by stimulation that activates the hair cell receptor conductance. The relation between receptor current and transmitter release cannot be studied at the intact semicircular canal. To circumvent the problem, we combined patch-clamp recordings at the isolated hair cell and electrophysiological recordings at the cytoneural junction in the intact preparation. At isolated hair cells, the K channel blocker tetraethylammonium (TEA) is shown to block a fraction of total voltage-dependent K-conductance (IKD) that depends on TEA concentration but not on membrane potential (V m). Considering the bioelectric properties of the hair cell, as previously characterized by this lab, a fixed fractional block of IKD is shown to induce a relatively fixed shift in V m, provided it lies in the range -30 to -10 mV. The same concentrations of TEA were applied to the intact labyrinth while recording from single afferent fibers of the posterior canal, at rest and during mechanical stimulation. At the peak of stimulation, TEA produced increases in mEPSP rate that were linearly related to the shifts produced by the same TEA concentrations (0.1-3 mM) in hair cell V m (0.7-5 mV), with a slope of 29.8 Hz/mV. The membrane potential of the hair cell is not linearly related to receptor conductance, so that the slope of quantal release vs. receptor conductance depends on the prevailing V m (19.8 Hz/nS at -20 mV; 11 Hz/nS at -10 mV). Changes in mEPSP peak size were negligible at rest as well as during stimulation. Since ample spatial summation of mEPSPs occurs at the afferent terminal and threshold-governed spike firing is intrinsically nonlinear, the observed increases in mEPSP frequency, though not very large, may suffice to trigger afferent spike discharge.

11.
PLoS One ; 8(7): e67784, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23844092

RESUMO

In hair cells dissected from the frog crista ampullaris, the combination of a calcium-dependent (IKCa) and a purely voltage-dependent component (IKV) gives rise to the delayed potassium current complex (IKD). These currents have been recently reported to display slow depolarization-induced inactivation and biphasic inactivation removal by hyperpolarization. The amplitude and inactivation kinetics of both IKCa and IKV are drastically modulated by a previously unrecognized mechanism of protein phosphorylation (sensitive to kinase inhibitors H89 and KT5823), which does not interfere with the transient potassium current (IA) or the calcium current (ICa). IKD amplitude was stable in cells patched with pipettes containing 8 mM ATP or under perforated-patch; under these conditions, a 10 min treatment with 10 µM H89 or 1-10 µM KT5823 reduced IKD amplitude by a mean of 67% at +40 mV. Similarly affected was the isolated IKV component (ICa blocked with Cd(2+)). Thus, a large potassium conductance can be activated by depolarization, but it is made available to the cell to a variable extent that depends on membrane potential and protein kinase activity. The total gKD ranged 4.6-44.0 nS in control cells, according to the level of steady-state inactivation, and was reduced to 1.4-2.7 nS after protein kinase inhibition. When sinusoidal membrane potential changes in the -70/-10 mV range were applied, to mimic receptor response to hair bundle deflection, IKD proved the main current dynamically activated and the only one regulated by PK: H89 decreased the total outward charge during each cycle by 60%. Phosphorylation appears to control both the amount of IKCa and IKV conductance activated by depolarization and the fraction thereof which can be rescued by removal of inactivation. The balance between the depolarizing transduction current and the repolarizing potassium current, and eventually the transmitter release at the cytoneural junction, are therefore modulated by a phosphorylation-mediated process.


Assuntos
Células Ciliadas Auditivas/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Proteínas Quinases/metabolismo , Rana esculenta/fisiologia , Canais Semicirculares/metabolismo , Animais , Cádmio/farmacologia , Cálcio/metabolismo , Carbazóis/farmacologia , Cátions Bivalentes , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Isoquinolinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Canais Semicirculares/citologia , Canais Semicirculares/efeitos dos fármacos , Sulfonamidas/farmacologia , Fatores de Tempo
12.
Hear Res ; 282(1-2): 151-60, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21906667

RESUMO

The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (∼34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics. The I(KCa) component of the delayed I(KD) was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca(2+) independent fraction of I(KD), I(KV), and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both I(Ca) and I(KD) amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of I(KD) inactivation, although the changes were scaled to the reduced I(KD) amplitude. From these observations, it is expected that hair cells exposed to gentamicin 'in vivo' become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca(2+) inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis.


Assuntos
Antibacterianos/toxicidade , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Gentamicinas/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Canais Semicirculares/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas/metabolismo , Transporte de Íons , Potenciais da Membrana , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Rana esculenta , Canais Semicirculares/citologia , Canais Semicirculares/metabolismo , Fatores de Tempo
14.
PLoS One ; 6(2): e17318, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21364885

RESUMO

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.


Assuntos
Cátions/metabolismo , Cloretos/fisiologia , Receptores Nicotínicos/metabolismo , Gânglio Cervical Superior/metabolismo , Sinapses/metabolismo , Acetilcolina/farmacologia , Animais , Bungarotoxinas/farmacologia , Células Cultivadas , Cloretos/metabolismo , Cloretos/farmacologia , Eletrofisiologia , Gânglios/efeitos dos fármacos , Gânglios/metabolismo , Gânglios/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Receptores Nicotínicos/fisiologia , Especificidade por Substrato , Gânglio Cervical Superior/fisiologia , Sinapses/fisiologia
15.
Am J Physiol Regul Integr Comp Physiol ; 296(5): R1585-97, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19244579

RESUMO

The effects of microgravity on the biophysical properties of frog labyrinthine hair cells have been examined by analyzing calcium and potassium currents in isolated cells by the patch-clamp technique. The entire, anesthetized frog was exposed to vector-free gravity in a random positioning machine (RPM) and the functional modification induced on single hair cells, dissected from the crista ampullaris, were subsequently studied in vitro. The major targets of microgravity exposure were the calcium/potassium current system and the kinetic mechanism of the fast transient potassium current, I(A). The amplitude of I(Ca) was significantly reduced in microgravity-conditioned cells. The delayed current, I(KD) (a complex of I(KV) and I(KCa)), was drastically reduced, mostly in its I(KCa) component. Microgravity also affected I(KD) kinetics by shifting the steady-state inactivation curve toward negative potentials and increasing the sensitivity of inactivation removal to voltage. As concerns the I(A), the I-V and steady-state inactivation curves were indistinguishable under normogravity or microgravity conditions; conversely, I(A) decay systematically displayed a two-exponential time course and longer time constants in microgravity, thus potentially providing a larger K(+) charge; furthermore, I(A) inactivation removal at -70 mV was slowed down. Stimulation in the RPM machine under normogravity conditions resulted in minor effects on I(KD) and, occasionally, incomplete I(A) inactivation at -40 mV. Reduced calcium influx and increased K(+) repolarizing charge, to variable extents depending on the history of membrane potential, constitute a likely cause for the failure in the afferent mEPSP discharge at the cytoneural junction observed in the intact labyrinth after microgravity conditioning.


Assuntos
Canais de Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Canais de Potássio/metabolismo , Rana esculenta/fisiologia , Canais Semicirculares/metabolismo , Ausência de Peso , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células Ciliadas Auditivas/citologia , Potenciais da Membrana/fisiologia , Modelos Animais , Técnicas de Patch-Clamp
16.
Pflugers Arch ; 457(6): 1327-42, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18936960

RESUMO

Potassium-current inactivation and recovery kinetics are pivotal in sustaining dynamic processing of time-varying sensory signals in hair cells. We report a detailed analysis of K(+)-currents in isolated hair cells from the frog crista ampullaris. The single components were dissected using a novel procedure based on their differential kinetic properties: The fast IA component exhibited two processes of inactivation removal; the persistent I (KD) component (I (KV) + I (KCa)), unexpectedly displayed partial inactivation, removed by negative potentials with particularly slow, delayed kinetics. The physiological relevance of these observations was investigated by imposing sinusoidal membrane potential changes to mimic receptor response to hair bundle deflection. The excitatory phase elicited extra-currents (hysteresis) only if the off phase went sufficiently negative to remove IA inactivation. Native, resting hair cells are depolarised by receptor current; thus, voltage continuously modulates I(KD), whereas IA only transiently ensues when the receptor current vanishes (zero-current potential approximately -70 mV) and polarisation removes IA inactivation.


Assuntos
Células Ciliadas Auditivas/fisiologia , Potenciais da Membrana/fisiologia , Canais de Potássio/fisiologia , Animais , Cádmio/farmacologia , Cálcio/fisiologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização , Rana esculenta
17.
Eur J Neurosci ; 25(4): 1112-26, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17331207

RESUMO

The mechanisms that control chloride conductance (gCl) in the rat sympathetic neuron have been studied by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. In addition to voltage dependence in the membrane potential range -120/-50 mV, gCl displays time- and activity-dependent regulation (sensitization). The resting membrane potential is governed by voltage-dependent gK and gCl, which determine values of cell input conductance ranging from 7 to 18 nS (full deactivation) to an upper value of about 130 nS (full activation and maximal gCl sensitization). The quiescent neuron, held at constant membrane potential, spontaneously and gradually moved from a low- to a high-conductance status. An increase (about 40 nS) in gCl accounted for this phenomenon, which could be prevented by imposing intermittent hyperpolarizing episodes. Following spike firing, gCl increased by 20-33 nS, independent of the cell conductance value preceding tetanization, and thereafter decayed to the pre-stimulus level within 5 min. Intracellular sodium depletion and its successive ionophoretic restoration moved the neuron from a stable low-conductance state to maximum gCl sensitization, pointing to a link between gCl sensitization and [Na+]i. The dependence of gCl build-up on [Na+]i and the time-course of such Na+-related modulation have been examined: gCl sensitization was absent at 0 [Na+]i, was well developed (20 nS) at 15 mM and tended towards a saturating value of 60 nS for higher [Na+]i. Sensitization was transient in response to neuron activity. In the silent neuron, sensitization of gCl shifted membrane potential over a range of about 15 mV.


Assuntos
Canais de Cloreto/fisiologia , Cloretos/metabolismo , Neurônios/fisiologia , Gânglio Cervical Superior/citologia , Animais , Canais de Cloreto/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Condutividade Elétrica , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Furosemida/farmacologia , Técnicas In Vitro , Eletrodos Seletivos de Íons , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Ouabaína/farmacologia , Técnicas de Patch-Clamp/métodos , Ratos , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Fatores de Tempo
18.
J Neurophysiol ; 95(5): 2832-44, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16452258

RESUMO

A biophysical description of the axotomized rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. Multiple aspects of neuron functioning were tested. Synaptic conductance activated by the whole presynaptic input decreased to 29% of the control value (0.92 muS per neuron) 1 day after axotomy and to 18% after 3 days. Despite the decrease in amplitude of the macroscopic current, miniature excitatory postsynaptic current (mEPSC) mean conductance, acetylcholine (ACh) equilibrium potential, and EPSC decay time constant were unaffected. Synaptic efficacy was tested during paired-pulse or maintained stimulation (5, 10, and 15 Hz, 10-s duration). Quantal release in axotomized neurons was preserved during the tetanus despite the reduction of the initial EPSC amplitude, suggesting that ACh secretion depended on the number of surviving synapses; each of them exhibited dynamic behavior during trains similar to that of normal synapses. Facilitation of EPSC amplitude was noted in 2-day axotomized neurons during the first few impulses in the train. Voltage-dependent potassium currents (the delayed I(KD) and the transient I(A)) exhibited an early drastic decrease in peak amplitude; these effects persisted 7 days after axotomy. Marked changes in I(A) kinetics occurred after injury: the steady-state inactivation curve shifted by up to +17 mV toward positive potentials and the voltage sensitivity of inactivation removal became steeper. I(A) impairment was reflected in a reduced inward threshold charge for discharge and reduced spike repolarization rate. Synaptic and somatic data were applied in a mathematical model to describe the progressive decrease in the safety factor, and the eventual failure of ganglionic transmission after axotomy.


Assuntos
Axotomia , Lateralidade Funcional/fisiologia , Gânglios Simpáticos/citologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Potássio/farmacologia , Ratos , Fatores de Tempo
19.
J Neurophysiol ; 90(2): 712-22, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12711705

RESUMO

Remarkable activity dependence was uncovered in the chloride conductance that operates in the subthreshold region of membrane potential, by using the two-microelectrode voltage-clamp technique in the mature and intact rat sympathetic neuron. Both direct and synaptic neuron tetanization (15 Hz, 10-s duration to saturate the response) resulted in a long-lasting (not less than 15 min) increase of cell input conductance (+70-150% 10 min after tetanus), accompanied by the onset of an inward current with the same time course. Both processes developed with similar properties in the postganglionic neuron when presynaptic stimulation was performed under current- or voltage-clamp conditions and were unaffected by external calcium on direct stimulation. The posttetanic effects were sustained by gCl increase because both conductance and current modifications were blocked by 0.5 mM Anthracene-9-carboxylic acid (a chloride channel blocker) but were unaffected by TEACl or cesium chloride treatments. The chloride channel properties were modified by stimulation: their voltage sensitivity and rate of closure in response to hyperpolarization strongly increased. The voltage dependence of the three major conductances governing the cell subthreshold status (gCl, gK, and gL) was evaluated over the -40/-110 mV membrane potential range in unstimulated neurons and compared with previous results in stimulated neurons. A drastic difference between the voltage-conductance profiles was observed, exclusively sustained by gCl increase. The chloride channel thus hosts an intrinsic mechanism, a memory of previous neuron activity, which makes the chloride current a likely candidate for natural controller of the balance between opposite resting currents and thus of membrane potential level.


Assuntos
Canais de Cloreto/fisiologia , Neurônios/fisiologia , Gânglio Cervical Superior/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Cálcio/fisiologia , Estimulação Elétrica , Feminino , Masculino , Potenciais da Membrana , Modelos Neurológicos , Técnicas de Patch-Clamp , Ratos
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