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/farmacologiaRESUMO
Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling-induced chloride current ICl,swell . In this study, we investigated the effects of ICl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of ICl,swell , DCPIB, potently reduced the ICl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB-treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the ICl,swell may be a potential drug target for GBM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Ciclopentanos/farmacologia , Glioblastoma/tratamento farmacológico , Indanos/farmacologia , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Canais de Cloreto/metabolismo , Relação Dose-Resposta a Droga , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Janus Quinase 2/metabolismo , Potenciais da Membrana , Invasividade Neoplásica , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND/AIMS: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. METHODS: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. RESULTS: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by â¼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. CONCLUSION: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Tamanho Celular/efeitos dos fármacos , Glicina/farmacologia , Sistema A de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Cloretos/metabolismo , Ciclopentanos/farmacologia , Soluções Hipotônicas/farmacologia , Indanos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microglia/citologia , Microglia/metabolismo , Nitrobenzoatos/farmacologia , Técnicas de Patch-ClampRESUMO
BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Animais , Cloretos/metabolismo , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos , Células NIH 3T3 , Ácido Niflúmico/química , Ácido Niflúmico/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
ß-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether ß-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of ß-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. ß-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately -70â¯mV, which was close to the equilibrium potential of Cl-. Pharmacological analysis revealed that ß-alanine activates glycine receptors, but not GABAA receptors. These results suggest that ß-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl- channels through glycine receptors. Our findings raise the possibility that ß-alanine may modulate pain sensation through glycine receptors.
Assuntos
Nociceptividade/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Receptores de Glicina/metabolismo , Substância Gelatinosa/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , beta-Alanina/farmacologia , Animais , Glicina/metabolismo , Glicina/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Nociceptividade/fisiologia , Técnicas de Patch-Clamp , Células do Corno Posterior/citologia , Células do Corno Posterior/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Receptores de Glicina/agonistas , Substância Gelatinosa/citologia , Substância Gelatinosa/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
BACKGROUND: Although pharmacological treatment has increased the average life expectancy of patients with cystic fibrosis, the median survival of females is shorter than that of males. In vitro and in vivo studies have shown that estrogens play a relevant role in the disease progression. The aim of this study was to investigate the effects of 17ß-estradiol and tamoxifen citrate (TMX) on calcium-activated chloride channel (CaCC) currents in human bronchial epithelial cells carrying the ΔPhe508-CFTR mutation both in homozygosis and in heterozygosis. METHODS: Perforated patch clamp experiments were performed on single cells of the immortalized cell lines CFBE and IB3-1. Gramicidin (10 or 20 µM) was added to the electrode solution to reach the whole cell configuration. The electrical stimulation protocol consisted of square voltages ranging from - 80 to + 80 mV, in steps of 20 mV and with a duration of 800 msec. RESULTS: The presence of 17ß-estradiol significantly reduced the CaCC currents, both in basal conditions and in the presence of ATP (100 µM). The addition of TMX (10 µM) completely restored the currents abolished by 17ß-estradiol, in basal conditions and after stimulation with ATP in both CFBE and IB3-1 cells. TMX had a strong, direct action on membrane current density, which significantly increased more than 4-fold in both cases. The membrane current stimulation produced by TMX was further enhanced by the addition of ATP. CFBE cells incubated for 24 h with 3 µM VX-809 (a CFTR corrector) and then acutely stimulated with VX-770 (a CFTR potentiator) in the presence of forskolin, showed an increase of chloride currents which were abolished by Inh-172. The chloride current density induced by TMX + ATP was, on average, greater than that obtained with VX-809 + VX-770 + forskolin. The currents elicited by TMX + ATP were abolished by the addition of NPPB, a CaCC inhibitor. The combined administration of TMX/ATP and VXs/FSK had an additional effect on chloride currents. CONCLUSIONS: Our results show that TMX restores CaCC currents inhibited by 17ß-estradiol and directly activates the transmembrane chloride currents potentiated by ATP, an effect which is mutation independent. The combined effect of TMX with current used treatments for cystic fibrosis could be of benefit to patients.
Assuntos
Canais de Cloreto/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Moduladores de Receptor Estrogênico/farmacologia , Mutação Puntual/genética , Mucosa Respiratória/efeitos dos fármacos , Tamoxifeno/farmacologia , Linhagem Celular Transformada , Canais de Cloreto/fisiologia , Estradiol/farmacologia , Humanos , Mucosa Respiratória/fisiologiaRESUMO
Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 µM) reduced IClCa by ~85%, while 30 µM abolished it. L-type Ca2+ current was unaffected by 10 µM CaCCinh-A01 but was reduced by 50% at 30 µM CaCCinh-A01, 46% at 10 µM T16Ainh-A01, and 78% at 30 µM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 µM and >90% at 30 µM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 µM, CaCCinh-A01; 14 µM, T16Ainh-A01). CaCCinh-A01 at 10 µM had little effect on 60 mM KCl contractures, though they were reduced by 30 µM CaCCinh-A01 and T16Ainh-A01 (10 µM and 30 µM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 µM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Músculo Liso/fisiologia , Pênis/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Pênis/efeitos dos fármacos , CoelhosRESUMO
Ca2+ signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+ , there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store-operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células Acinares/metabolismo , Animais , Humanos , Oócitos/metabolismoRESUMO
Thiazides block Na+ reabsorption while enhancing Ca2+ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca2+ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl- channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl- channels, however the nature of these Cl- channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl- current at extracellular pH7.4. This constitutive Cl- current was more permeable to larger anions (Eisenman sequence I; I->Br-≥Cl-) and was substantially inhibited by >100mM [Ca2+]o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl- current was blocked by NPPB, along with other Cl- channel inhibitors (4,4'-diisothiocyanatostilbene-2,2'-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH<5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl- current. This acid-induced Cl- current was also anion permeable (I->Br->Cl-), but was distinguished from the constitutive Cl- current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl- current would be particularly relevant in the acidic IMCD (pH<5.5). To our knowledge, the properties of these Cl- currents are unique and provide the mechanisms to account for the Cl- efflux previously speculated to be present in MDCT cells.
Assuntos
Canais de Cloreto/fisiologia , Túbulos Renais Distais/metabolismo , Animais , Células Cultivadas , Cloretos/metabolismo , Concentração de Íons de Hidrogênio , CamundongosRESUMO
Striatal low-threshold spiking (LTS) interneurons spontaneously transition to a depolarized, oscillating state similar to that seen after sodium channels are blocked. In the depolarized state, whether spontaneous or induced by sodium channel blockade, the neurons express a 3- to 7-Hz oscillation and membrane impedance resonance in the same frequency range. The membrane potential oscillation and membrane resonance are expressed in the same voltage range (greater than -40 mV). We identified and recorded from LTS interneurons in striatal slices from a mouse that expressed green fluorescent protein under the control of the neuropeptide Y promoter. The membrane potential oscillation depended on voltage-gated calcium channels. Antagonism of L-type calcium currents (CaV1) reduced the amplitude of the oscillation, whereas blockade of N-type calcium currents (CaV2.2) reduced the frequency. Both calcium sources activate a calcium-activated chloride current (CaCC), the blockade of which abolished the oscillation. The blocking of any of these three channels abolished the membrane resonance. Immunohistochemical staining indicated anoctamin 2 (ANO2), and not ANO1, as the CaCC source. Biophysical modeling showed that CaV1, CaV2.2, and ANO2 are sufficient to generate a membrane potential oscillation and membrane resonance, similar to that in LTS interneurons. LTS interneurons exhibit a membrane potential oscillation and membrane resonance that are both generated by CaV1 and CaV2.2 activating ANO2. They can spontaneously enter a state in which the membrane potential oscillation dominates the physiological properties of the neuron.
Assuntos
Corpo Estriado/fisiologia , Interneurônios/metabolismo , Canais Iônicos/metabolismo , Potenciais da Membrana/fisiologia , Animais , Bloqueadores dos Canais de Cálcio , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Camundongos Transgênicos , Modelos Moleculares , Modelos Neurológicos , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Periodicidade , Regiões Promotoras Genéticas , Técnicas de Cultura de TecidosRESUMO
During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation. Using a combination of electrophysiology and immunohistochemistry, we found that the chloride intracellular channel 1 (CLIC1) protein modulates the speed of neurite growth. The chloride current mediated by CLIC1 is essential for maintaining growth cone morphology and is required for sustaining cAMP-stimulated neurite elongation in dissociated immunopurified neurons. The presence of either the CLIC1 current blocker IAA94 or the anti-CLIC1 antibody inhibits neurite growth of Retina Ganglion Cells cultured in the presence of 10 micromolar forskolin for 24 h.
Assuntos
Canais de Cloreto/metabolismo , AMP Cíclico/farmacologia , Neuritos/efeitos dos fármacos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/imunologia , Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , Glicolatos/farmacologia , Hipocampo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Técnicas de Patch-Clamp , Retina/citologiaRESUMO
INTRODUCTION: Although adenosine triphosphate (ATP) has often been reported to relax the corpus cavernosum, this may be mediated by indirect effects, such as release of nitric oxide from the endothelium. Recent data suggest that P2X(1) receptors may be up-regulated in diabetes, and these exert an anti-erectile effect by causing the corpus cavernosum smooth muscle cells (CCSMCs) to contract. However, to date, there is no functional evidence that ATP can directly stimulate CCSMC. AIMS: This study aims to (i) to directly examine the effect of ATP on membrane currents in freshly isolated CCSMC, where influences of endothelium and other cells are absent; and (ii) to determine the receptor subtypes, ionic currents, and Ca(2+) signals stimulated by ATP. METHODS: CCSMCs were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and measurement of intracellular Ca(2+) in fluo-4-loaded cells using spinning disk confocal microscopy. MAIN OUTCOME MEASURES: Patch clamp recordings were made of ATP-evoked membrane currents and spontaneous membrane currents. Spinning disk confocal imaging of intracellular Ca(2+) was performed, and the response to ATP was recorded. RESULTS: ATP evoked repeatable inward currents in CCSMC (1st application: -675 ± 101 pA; 2nd application: -694 ± 120 pA, N = 9, P = 0.77). ATP-induced currents were reduced by suramin from -380 ± 121 to -124 ± 37 pA (N = 8, P < 0.05), by α,ß-methylene ATP from -755 ± 235 to 139 ± 49 pA (N = 5, P < 0.05), and by NF449 from -419 ± to -51 ± 13 pA (N = 6, P < 0.05). In contrast, MRS2500, a P2Y1(1,12,13) antagonist, had no effect on ATP responses (control: -838 ± 139 pA; in MRS2500: -822 ± 184 pA, N = 13, P = 0.84) but blocked inward currents evoked by 2-MeSATP, a P2Y1,12,13 agonist (control: -623 ± 166 pA; in MRS2500: -56 ± 25 pA, N = 6, P < 0.05). The ATP-evoked inward current was unaffected by changing the transmembrane Cl(-) gradient but reversed in direction when extracellular Na(+) was reduced, indicating that it was a cation current. CONCLUSIONS: ATP directly stimulates CCSMC by evoking a P2X-mediated cation current.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Células Musculares/efeitos dos fármacos , Pênis/fisiologia , Tionucleotídeos/farmacologia , Trifosfato de Adenosina/farmacologia , Compostos de Anilina/análise , Animais , Cálcio/farmacologia , Cálcio/fisiologia , Masculino , Células Musculares/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ereção Peniana/efeitos dos fármacos , Ereção Peniana/fisiologia , Coelhos , Xantenos/análiseRESUMO
We examined the effects of a dihydropyridine analog, PAK-200, on guinea pig myocardium during experimental ischemia and reperfusion. In isolated ventricular cardiomyocytes, PAK-200 (1 µM) had no effect on the basal peak inward and steady-state currents but inhibited the isoprenaline-induced time-independent Cl- current. In the right atria, PAK-200 had no effect on the beating rate and the chronotropic response to isoprenaline. In an ischemia-reperfusion model with coronary-perfused right ventricular tissue, a decrease in contractile force and a rise in tension were observed during a period of 30-min no-flow ischemia. Upon reperfusion, contractile force returned to less than 50% of preischemic values. PAK-200 had no effect on the decline in contractile force during the no-flow ischemia but reduced the rise in resting tension. PAK-200 significantly improved the recovery of contractile force after reperfusion to about 70% of the preischemic value. PAK-200 was also shown to attenuate the decrease in tissue ATP during ischemia. Treatment of ventricular myocytes with an ischemia-mimetic solution resulted in depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca2+ concentrations. PAK-200 significantly delayed these changes. Thus, PAK-200 inhibits the cAMP-activated chloride current in cardiac muscle and may have protective effects against ischemia-reperfusion injury through novel mechanisms.
Assuntos
Di-Hidropiridinas , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Cobaias , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Isoproterenol/farmacologia , Cloretos/farmacologia , Di-Hidropiridinas/farmacologia , Isquemia , Miócitos Cardíacos , Contração MiocárdicaRESUMO
Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl- currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.
RESUMO
Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9'-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.
RESUMO
Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Antracenos/farmacologia , Cloretos/agonistas , Cloretos/antagonistas & inibidores , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Humanos , Soluções Hipotônicas/metabolismo , Soluções Hipotônicas/farmacologia , Indóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Maleimidas/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Dibutirato de 12,13-Forbol/farmacologia , Cultura Primária de CélulasRESUMO
An ethyl acetate extract of Curcuma kwangsiensis S.G. Lee & C.F. Liang (Zingiberaceae) rhizomes (100 µg/ml) enhanced the GABA-induced chloride current (IGABA) through GABAA receptors of the α1ß2γ2S subtype by 79.0±7.0%. Potentiation of IGABA was measured using the two-microelectrode voltage-clamp technique and Xenopus laevis oocytes. HPLC-based activity profiling of the crude extract led to the identification of 11 structurally related labdane diterpenoids, including four new compounds. Structure elucidation was achieved by comprehensive analysis of on-line (LC-PDA-ESI-TOF-MS) and off-line (microprobe 1D and 2D NMR) spectroscopic data. The absolute configuration of the compounds was established by comparison of experimental and calculated ECD spectra. Labdane diterpenes represent a new class of plant secondary metabolites eliciting positive GABAA receptor modulation. The highest efficiency was observed for zerumin A (maximum potentiation of IGABA by 309.4±35.6%, and EC50 of 24.9±8.8 µM).
Assuntos
Curcuma/química , Diterpenos/isolamento & purificação , Diterpenos/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Diterpenos/química , Medicamentos de Ervas Chinesas/química , Ressonância Magnética Nuclear Biomolecular , Oócitos/metabolismo , Rizoma/química , Xenopus/embriologiaRESUMO
Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Assuntos
Humanos , Antracenos , Farmacologia , Canais de Cloreto , Metabolismo , Cloretos , Metabolismo , Meios de Cultura , Metabolismo , Farmacologia , Relação Dose-Resposta a Droga , Potenciais Evocados , Fisiologia , Átrios do Coração , Biologia Celular , Metabolismo , Soluções Hipotônicas , Metabolismo , Farmacologia , Indóis , Farmacologia , Transporte de Íons , Maleimidas , Farmacologia , Miócitos Cardíacos , Biologia Celular , Metabolismo , Técnicas de Patch-Clamp , Dibutirato de 12,13-Forbol , Farmacologia , Cultura Primária de Células , Proteína Quinase C , MetabolismoRESUMO
The whole-cell patch clamp technique and "Y-tube" method were applied to evaluate the effects of sevoflurane (from 0. 3×10-3to 3×10-3 mol/L) on chloride current induced by bath utilization of 3×10-6 mol/L γ-aminobutyric acid (GABA) in the single-cultured rat dorsal root ganglia neurons. Experimental data demonstrated that when peak amplitude of chloride current induced by 3 × 10-6 mol/L GABA was considered as 100 % in the presence of 0. 38×10-3, 0. 76×10-3, 2. 28×10-3, 3. 04×10 -3 mol/L sevoflurane peak amplitude of chloride current was rose to (149±25) %, (203±-27) %, (327±79) %, (331±109) %, (243±71) % correspondingly. This finding suggests that sevoflurane, at concentrations relevant clinical anesthesia, can enhance GABA-mediated chloride current in sensory neurons.