RESUMEN
Calcium-activated chloride channels (CaCCs) are thought to regulate neuronal excitability, and recently chloride (Cl(-)) regulation in DRG neurons has attracted much attention in pain research. Furthermore, the activity of CaCCs is modified by a family of CLCA proteins. In acute antigen-induced arthritis (AIA), a remarkable up-regulation of the murine chloride channel accessory 3 (mClca3) was shown in dorsal root ganglion (DRG) neurons. Therefore we tested the hypothesis that mClca3 is involved in arthritic pain perception. In mClca3 knock-out mice and wild-type control mice, AIA was induced and measures of inflammation and pain were assessed. In the very acute phase of AIA, joint swelling was reduced in mClca3 knock-out mice. This effect disappeared during the course of AIA. We could not show significant differences in mechanical hyperalgesia between both groups of mice, neither at the acute nor at the chronic stage (21 days of AIA). Additional experiments on thermal hyperalgesia in wild-type and mClca3 knock-out mice in the first 3 days of AIA did not show a difference either. In addition, niflumic acid, an antagonist at CaCCs, did not significantly influence hyperalgesia during AIA. Thus, we were not able to provide evidence for a role of CaCCs, and in particular of mClca3, on the expression of arthritis or inflammation-evoked hyperalgesia.
Asunto(s)
Artritis/metabolismo , Canales de Cloruro/metabolismo , Mucoproteínas/metabolismo , Dolor/metabolismo , Animales , Artritis/fisiopatología , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Adyuvante de Freund , Ganglios Espinales/metabolismo , Calor , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Ratones Noqueados , Mucoproteínas/antagonistas & inhibidores , Mucoproteínas/genética , Neuronas/metabolismo , Ácido Niflúmico/farmacología , Dolor/fisiopatología , Dolor/psicología , Percepción del DolorRESUMEN
1. 1,8-Cineole is a non-toxic small terpenoid oxide believed to have medicinal properties in folk medicine. It has been shown to have various pharmacological effects, including blockade of the compound action potential (AP). In the present study, using intracellular recording techniques, we investigated the effects of 1,8-cineole on the electrophysiological parameters of neurons of the superior cervical ganglion (SCG) in rats. 2. 1,8-Cineole (0.1-6 mmol/L) showed reversible and concentration-dependent effects on various electrophysiological parameters. At 3 and 6 mmol/L, but not at 0.1 and 1 mmol/L, 1,8-cineole significantly diminished the input resistance (R(i)) and altered the resting potential (E(m)) to more positive values. At 6 mmol/L, 1,8-cineole completely blocked all APs within 2.7 +/- 0.6 min (n = 12). In neurons exposed to 3 and 1 mmol/L 1,8-cineole, the effects regarding excitability varied from complete AP blockade to minor inhibition of AP parameters. The depolarization of E(m) and the decrease in R(i) induced by 6 mmol/L 1,8-cineole were unaltered by 200 micromol/L niflumic acid, a well known blocker of Ca(2+)-activated Cl(-) currents. 3. Significant correlations (Pearson correlation test) were found between changes in E(m) and decreases in AP amplitude (r = -0.893; P < 0.00282) and maximum ascendant inclination (r = -0.799; P < 0.0173), but not for maximum descendant inclination (r = 0.598; P < 0.117). Application of current to restore the transmembrane potential equal to control E(m) values in the presence of 6 mmol/L 1,8-cineole resulted in the partial recovery of AP. 4. The present study shows that 1,8-cineole effectively blocks the excitability of SCG neurons, probably through various mechanisms, one of which acts indirectly via depolarization of the neuronal cytoplasmatic membrane.
Asunto(s)
Ciclohexanoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Monoterpenos/farmacología , Neuronas/fisiología , Ganglio Cervical Superior/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Eucaliptol , Femenino , Masculino , Medicina Tradicional , Ácido Niflúmico/farmacología , Ratas , Ratas WistarRESUMEN
Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway.
Asunto(s)
Canales de Cloruro/fisiología , Colon/metabolismo , Emodina/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Calcio/metabolismo , Colon/citología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Motilidad Gastrointestinal/efectos de los fármacos , Cobayas , Técnicas In Vitro , Masculino , Miocitos del Músculo Liso/metabolismo , Ácido Niflúmico/farmacología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca2+-free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-beta-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca2+ influx, phospholipase C activation, and Ca2+ release from internal storage in a G protein-independent and protein kinase C-independent manner.
Asunto(s)
Intestino Delgado/efectos de los fármacos , Preparaciones de Plantas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Calcio/metabolismo , Femenino , Ácido Flufenámico/farmacología , Fármacos Gastrointestinales/farmacología , Motilidad Gastrointestinal/fisiología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ácido Niflúmico/farmacología , Técnicas de Placa-Clamp , Fosfolipasas de Tipo C/metabolismoRESUMEN
We previously reported the cloning of a calcium-activated chloride channel (CLCA) from rat brain (Biochem. Biophys. Res. Commun., 334, 569-576 (2005)), which we designated rbCLCA1. We further showed that rbCLCA1 is expressed in the central nervous system and peripheral organs, and may be functionally expressed in mammalian HEK293 cells. In the present study, we report the successful cloning of a second CLCA from rat cerebrum (designated rbCLCA2), using reverse transcription-PCR (RT-PCR) with primers specific for rbCLCA1. We have begun to clone this cDNA based on the rbCLCA1-like sequence. The full-length rbCLCA2 cDNA, obtained via 5' and 3' rapid amplification of cDNA ends (RACE), is 2900 bp long and encodes a putative polypeptide of 905 amino acids having at least two major transmembrane domains and showing 85.2% identity to rbCLCA1. RT-PCR analysis revealed that, similar to rbCLCA1, rbCLCA2 was predominantly expressed in the rat cerebrum, cerebellum, kidney, stomach, spinal cord, lung and small intestine, but not in the heart, large intestine, liver, orand spleen. Whole-cell patch clamp studies in HEK293 cells transiently co-transfected with expression vectors encoding rbCLCA2 and EGFP allowed us to identify the presence of niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride currents in cells expressing rbCLCA2 but not EGFP alone. Treatment of these cells with ionomycin, a Ca2+ ionophore, significantly increased the novel currents in cells expressing rbCLCA2 and EGFP, but not those expressing EGFP alone, indicating that activation of the rbCLCA2 current is Ca2+-dependent. In sum, we herein report the cloning of a second member of the rbCLCA family from rat brain and its functional expression in vitro, thus adding to our knowledge of anion channels and facilitating future exploration of brain and other organ physiology.
Asunto(s)
Encéfalo/metabolismo , Canales de Cloruro/genética , Expresión Génica/genética , Secuencia de Aminoácidos , Animales , Cloruro de Calcio/farmacología , Línea Celular , Canales de Cloruro/metabolismo , Canales de Cloruro/fisiología , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Ácido Egtácico/farmacología , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Riñón/citología , Riñón/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Ácido Niflúmico/farmacología , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transfección/métodosRESUMEN
Taste bud cells are epithelial cells with neuronal properties. Voltage-dependent ion channels have been physiologically described in these cells. Here, we report the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. ClC-4A skipped an exon near its 5'-end, incurring the loss of 60 amino acids at the N terminus. In situ hybridization and immunohistochemistry localized these two channels' transcripts and proteins to a subset of taste bud cells. Electrophysiological recordings of the heterologously expressed channels in Xenopus oocytes showed that ClC-4 and ClC-4A have opposite sensitivity to pH and unique ion selectivity. The chloride channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid had a slight or no inhibitory effect on the conductance of ClC-4, but both blockers inhibited ClC-4A, suggesting that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive current. Furthermore, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles.
Asunto(s)
Empalme Alternativo , Canales de Cloruro/genética , Papilas Gustativas/patología , Inhibidores de la Angiogénesis/farmacología , Animales , Canales de Cloruro/fisiología , Inhibidores de la Ciclooxigenasa/farmacología , ADN Complementario/metabolismo , Electrofisiología , Exones , Humanos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Hibridación in Situ , Iones , Ratones , Datos de Secuencia Molecular , Ácido Niflúmico/metabolismo , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Hibridación de Ácido Nucleico , Oocitos/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Vesículas Sinápticas/metabolismo , XenopusRESUMEN
The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Germinación/efectos de los fármacos , Nicotiana/fisiología , Polen/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Antiportadores/efectos de los fármacos , Barbitúricos/farmacología , Bumetanida/farmacología , Furosemida/farmacología , Transporte Iónico/efectos de los fármacos , Isoxazoles/farmacología , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacologíaRESUMEN
Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.
Asunto(s)
Cloruros/metabolismo , Fosfatos de Inositol/farmacología , Polen/crecimiento & desarrollo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Canales de Cloruro/metabolismo , Lilium/citología , Lilium/efectos de los fármacos , Lilium/crecimiento & desarrollo , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Polen/efectos de los fármacos , Polen/metabolismo , Nicotiana/citología , Nicotiana/efectos de los fármacos , Nicotiana/crecimiento & desarrolloRESUMEN
Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.
Asunto(s)
Fabaceae/genética , Fabaceae/fisiología , Regulación de la Expresión Génica de las Plantas , Plantas Medicinales , Potasio/metabolismo , Antracenos/farmacología , Células Cultivadas , Medios de Cultivo , Inhibidores Enzimáticos/farmacología , Fabaceae/citología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Canales Iónicos/antagonistas & inhibidores , Cinética , Neomicina/farmacología , Ácido Niflúmico/farmacología , Péptidos , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/fisiología , Estaurosporina/farmacología , Superóxidos/metabolismo , Suramina/farmacología , Activación Transcripcional , Verapamilo/farmacología , Venenos de Avispas/farmacologíaRESUMEN
Calcium-activated Cl(-) secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca(2+)-stimulated Cl(-) channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X(7) purinoreceptor (P2X(7)-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca(2+)-activated Cl(-) current. In P2X(7)-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl(-) current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl(-) > I(-) > Br(-) > gluconate(-). We have also determined a rank order of potency for putative Cl(-) channel blockers: niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4, 4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients.
Asunto(s)
Calcio/metabolismo , Calixarenos , Cloruros/metabolismo , Receptores Purinérgicos P2/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Bromo/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Membrana Celular/metabolismo , Quelantes/farmacología , Fibrosis Quística/metabolismo , ADN Complementario/metabolismo , Electrofisiología , Epitelio/metabolismo , Epitelio/fisiología , Gluconatos/metabolismo , Gliburida/farmacología , Hipoglucemiantes/farmacología , Yodo/metabolismo , Iones , Ratones , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Fenoles/farmacología , Receptores Purinérgicos P2X7 , Tamoxifeno/farmacología , Factores de Tiempo , Tráquea/metabolismo , Tráquea/fisiología , Uridina Trifosfato/metabolismo , ortoaminobenzoatos/farmacologíaRESUMEN
A light scattering-based amyloid fibril formation assay was employed to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. Twenty nine aromatic small molecules, some with homology to flufenamic acid (a known non-steroidal anti-inflammatory drug) were tested to identify important structural features for inhibitor efficacy. The results of these experiments and earlier data suggest that likely inhibitors will have aromatic-based structures with at least two aromatic rings. The ring or fused ring system occupying the outermost TTR binding pocket needs to be substituted with an acidic functional group (e.g. a carboxylic acid) to interact with complimentary charges in the TTR binding site. The promising TTR amyloid fibril inhibitors ranked in order of efficacy are: 2 > 4 approximately 7 > 3 > 9 > 6 > 21.
Asunto(s)
Amiloide/ultraestructura , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Ácido Flufenámico/química , Prealbúmina/antagonistas & inhibidores , Amiloide/efectos de los fármacos , Amiloide/metabolismo , Antiinflamatorios no Esteroideos/metabolismo , Sitios de Unión , Diflunisal/química , Diflunisal/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ácido Flufenámico/metabolismo , Ácido Flufenámico/farmacología , Humanos , Luz , Ácido Niflúmico/química , Ácido Niflúmico/farmacología , Prealbúmina/química , Prealbúmina/metabolismo , Dispersión de Radiación , Relación Estructura-Actividad , Sulindac/química , Sulindac/farmacología , Tolmetina/química , Tolmetina/farmacologíaRESUMEN
The direct electrophysiological characterization of sperm Ca(2+) channels has been precluded by their small size and flat shape. An alternative to study these channels is to use spermatogenic cells, the progenitors of sperm, which are larger and easier to patch-clamp. In mouse and rat, the only voltage-dependent Ca(2+) currents displayed by these cells are of the T type. Because compounds that block these currents inhibit the zona pellucida-induced Ca(2+) uptake and the sperm acrosome reaction (AR) at similar concentrations, it is likely that they are fundamental for this process. Recent single channel recordings in mouse sperm demonstrated the presence of a Cl(-) channel. This channel and the zona pellucida (ZP)-induced AR were inhibited by niflumic acid (NA), an anion channel blocker [Espinosa et al. (1998): FEBS Lett 426:47-51]. Because NA and other anion channel blockers modulate cationic channels as well, it became important to determine whether they affect the T-type Ca(2+) currents of spermatogenic cells. These currents were blocked in a voltage-dependent manner by NA, 1, 9-dideoxyforskolin (DDF), and 5-nitro-2-(3-phenylpropylamine)benzoic acid (NPPB). The IC(50) values at -20 mV were 43 microM for NA, 28 microM for DDF, and 15 microM for NPPB. Moreover, DDF partially inhibited the ZP-induced AR (40% at 1 microM) and NPPB displayed an IC(50) value of 6 microM for this reaction. These results suggest that NA and DDF do not inhibit the ZP-induced AR by blocking T-type Ca(2+) currents, while NPPB may do so. Interestingly 200 microM NA was basically unable to inhibit alpha1E Ca(2+) channels expressed in Xenopus oocytes, questioning that this alpha subunit codes for the T-type Ca(2+) channels present in spermatogenic cells. Evidence for the presence of alpha1C, alpha1G, and alpha1H in mouse pachytene spematocytes and in round and condensing spermatids is presented.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Reacción Acrosómica/fisiología , Animales , Secuencia de Bases , Canales de Calcio/genética , Canales de Calcio/metabolismo , Colforsina/análogos & derivados , Colforsina/farmacología , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Técnicas In Vitro , Masculino , Ratones , Datos de Secuencia Molecular , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Ratas , Homología de Secuencia de Ácido Nucleico , Xenopus laevis , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.
Asunto(s)
Canales de Calcio/fisiología , Canales de Cloruro , Pulmón/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Bovinos , Línea Celular Transformada , ADN Complementario , Ditiotreitol/farmacología , Embrión de Mamíferos , Biblioteca de Genes , Humanos , Ionomicina/farmacología , Riñón , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Ácido Niflúmico/farmacología , Técnicas de Placa-Clamp , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , TransfecciónRESUMEN
Hypo-osmotic shock of aequorin-transformed tobacco cells induces a biphasic cytosolic Ca2+ influx. Because both phases of Ca2+ entry are readily blocked by Ca2+ channel inhibitors, we conclude that the Ca2+ transients are mediated by Ca2+ channels. Evidence that the first but not second Ca2+ transient derives from external Ca2+ stores is that the first but not second influx is (i) eliminated by membrane-impermeable Ca2+ chelators, (ii) enlarged by supplementation of the medium with excess Ca2+, and (iii) reduced by the addition of competitive cations such as Mg2+ and Mn2+. Furthermore, entry of 45Ca during osmotic shock is prevented by inhibitors of the first but not second phase of Ca2+ entry. Evidence that the second wave of Ca2+ influx stems from release of intracellular Ca2+ is based on the above data plus observations that probable modulators of intracellular Ca2+ channels selectively block this phase of Ca2+ influx. Finally, a mechanism of communication between the two Ca2+ release pathways has become apparent, since perturbations that elevate or reduce the first Ca2+ transient lead to a compensating diminution/elevation of the second and vice versa. These data thus suggest that osmotic shock leads to the sequential opening of extracellular followed by intracellular Ca2+ stores and that these Ca2+ release pathways are internally compensated.
Asunto(s)
Calcio/metabolismo , Nicotiana/metabolismo , Plantas Tóxicas , Aequorina , Transporte Biológico , Cafeína/farmacología , Canales de Calcio/metabolismo , Señalización del Calcio , Compartimento Celular , Citosol/metabolismo , Ácido Egtácico/farmacología , Canales Iónicos/metabolismo , Cinética , Lantano/farmacología , Magnesio/farmacología , Ácido Niflúmico/farmacología , Presión Osmótica , Estallido Respiratorio , Nicotiana/citologíaRESUMEN
cRNA encoding the human IsK protein was injected into Xenopus oocytes and the induced IsK channels were investigated using the two-microelectrode voltage-clamp method. Niflumic acid, mefenamic acid, flufenamic acid, and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, which are commonly used in Xenopus oocytes to suppress endogenous Ca(2+)-activated Cl- channels, were tested for their effects on IsK channels. At low concentrations (10 microM) all compounds increased IsK amplitude and decreased the rate of IsK deactivation. At 100 microM these compounds further decreased the rate of IsK deactivation, resulting in persistent activation of IsK, similar to what has been previously described for the action of organic cross-linkers on IsK. However, at 100 microM niflumic acid and flufenamic acid decreased the time-dependent outward current, whereas 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and mefenamic acid caused an additional increase. When Cl- was completely substituted with gluconate, IsK had somewhat altered activation properties, but niflumic acid produced similar positive regulatory effects on IsK and shifted the voltage needed to evoke half-maximal IsK activation (V1/2) by about -20 mV. In summary, these compounds positively regulate IsK, presumably by stabilizing open IsK channels.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Calcio/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Ácido Flufenámico/farmacología , Humanos , Ácido Mefenámico/farmacología , Potenciales de la Membrana , Ácido Niflúmico/farmacología , Oocitos , Canales de Potasio/genética , Canales de Potasio/fisiología , ARN Complementario , Proteínas Recombinantes , XenopusRESUMEN
Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.
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Fabaceae/fisiología , Canales Iónicos/fisiología , Plantas Medicinales , Benzoatos/farmacología , Ácido Benzoico , Western Blotting , Membrana Celular/fisiología , Ácido Etacrínico/farmacología , Fabaceae/citología , Glicolatos/farmacología , Canales Iónicos/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/fisiología , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Probenecid/farmacología , Protoplastos/fisiologíaRESUMEN
A hydroalcoholic extract of black currant (Ribes nigrum) leaves was tested on carrageenan-induced rat paw oedema. Pharmacological activity was compared with indomethacin and niflumic acid using acute and chronic (21 or 28 days) oral treatment. Black currant extract and lyophilisate revealed significant anti-inflammatory activity comparable to that seen with the reference substances, but without their ulcerogenic potential, even at high doses during chronic treatment.
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Antiinflamatorios no Esteroideos , Plantas Medicinales/análisis , Animales , Carragenina , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Francia , Mucosa Gástrica/efectos de los fármacos , Indometacina/farmacología , Masculino , Ácido Niflúmico/farmacología , Extractos Vegetales/efectos adversos , Extractos Vegetales/farmacología , Ratas , Ratas EndogámicasRESUMEN
Niflumic acid and indomethacin showed to be inhibitors of catecholamine--uptake into rat striatal and hypothalamic synaptosomes. Whereas the inhibition by niflumic acid was non-competitive for both amines at similar Ki values, the action of indomethacin displayed differences between areas as well as amines: the potency of inhibition by indomethacin was greater in striatal than in hypothalamic synaptosomes irrespective of the amine; the type of inhibition by indomethacin was non-competitive for norepinephrine, but competitive for dopamine regardless of the brain area.