RESUMEN
AIM: To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved and to establish whether activation leads to secretion of the neuropeptide calcitonin gene-related peptide (CGRP) and/or the translocation of NF-κB. METHODOLOGY: Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2 days. The effect of P. gingivalis LPS (20 µg mL-1 ) on calcium signalling was assessed (by calcium imaging using Cal-520 AM) in comparison with the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100 µmol L-1 ), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1 µmol L-1 ) and high potassium (60 mmol L-1 KCl). TG cultures were pre-treated with either 1 µmol L-1 CLI-095 to block Toll-like receptor 4 (TLR4) signalling or with 3 µmol L-1 HC-030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF-κB was investigated using immunocytochemistry. Data were analysed by one-way analysis of variance, followed by Bonferroni's post hoc test as appropriate. RESULTS: Porphyromonas gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non-neuronal cells (P < 0.001 to P < 0.05). The effects on neurons appear to be mediated via TLR4- and TRPA1-dependent pathways. The responses were accompanied by an increased release of CGRP (P < 0.001) and by NF-κB nuclear translocation (P < 0.01). CONCLUSIONS: Porphyromonas gingivalis LPS directly activated trigeminal sensory neurons (via TLR4 and TRPA1 receptors) and non-neuronal cells, resulting in CGRP release and NF-κB nuclear translocation. This indicates that P. gingivalis can directly influence activity in trigeminal sensory neurons and this may contribute to acute and chronic inflammatory pain.
Asunto(s)
Lipopolisacáridos , Porphyromonas gingivalis , Animales , Ratones , Dolor , Células Receptoras Sensoriales , Ganglio del TrigéminoRESUMEN
BACKGROUND: Prostaglandin E2 (PGE2 ) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2 -selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. OBJECTIVE: The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. METHODS: The effects of non-selective (PGE2 , misoprostol), EP2 -selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4 -selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2 -selective (PF-04418948, PF-04852946) and EP4 -selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. RESULTS: Prostaglandin E2 , EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4 -selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2 -selective antagonists caused rightward shifts in the PGE2 concentration-response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.
Asunto(s)
Dinoprostona/metabolismo , Liberación de Histamina/fisiología , Histamina/metabolismo , Inmunoglobulina E/metabolismo , Mastocitos/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Dinoprostona/agonistas , Dinoprostona/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Liberación de Histamina/efectos de los fármacos , Humanos , Pulmón , Mastocitos/citología , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidoresRESUMEN
BACKGROUND AND PURPOSE: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. KEY RESULTS: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 microM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by alphabetamethylene ATP (10 microM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 microM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 microM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 microM). P2X7-like responses were also evoked in LAD2 cells by 2'(3')-0-(4-benzoylbenzoyl)ATP (300 microM). A P2X4-like current was evoked by 100 microM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 microM). CONCLUSION AND IMPLICATIONS: Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.
Asunto(s)
Pulmón/metabolismo , Mastocitos/metabolismo , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/metabolismo , Cationes , Diferenciación Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Espacio Extracelular/metabolismo , Humanos , Canales Iónicos/fisiología , Mastocitos/citología , Técnicas de Placa-Clamp , ARN Mensajero/biosíntesis , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.
Asunto(s)
Actinas/metabolismo , Corteza Cerebral/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio , Corteza Cerebral/enzimología , Activación Enzimática , Gelsolina , Glucosidasas , Humanos , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismoRESUMEN
Angiotensin II (AngII) receptors couple to a multitude of different types of G-proteins resulting in activation of numerous signaling pathways. In this study we examined the consequences of this promiscuous G-protein coupling on secretion. Chromaffin cells were voltage-clamped at -80 mV in perforated-patch configuration, and Ca(2+)-dependent exocytosis was evoked with brief voltage steps to +20 mV. Vesicle fusion was monitored by changes in membrane capacitance (DeltaC(m)), and released catecholamine was detected with single-cell amperometry. Ca(2+) signaling was studied by recording voltage-dependent Ca(2+) currents (I(Ca)) and by measuring intracellular Ca(2+) ([Ca(2+)](i)) with fura-2 AM. AngII inhibited I(Ca) (IC(50) = 0.3 nm) in a voltage-dependent, pertussis toxin (PTX)-sensitive manner consistent with G(i/o)-protein coupling to Ca(2+) channels. DeltaC(m) was modulated bi-directionally; subnanomolar AngII inhibited depolarization-evoked exocytosis, whereas higher concentrations, in spite of I(Ca) inhibition, potentiated DeltaC(m) fivefold (EC(50) = 3.4 nm). Potentiation of exocytosis by AngII involved activation of phospholipase C (PLC) and Ca(2+) mobilization from internal stores. PTX treatment did not affect AngII-dependent Ca(2+) mobilization or facilitation of exocytosis. However, protein kinase C (PKC) inhibitors decreased the facilitatory effects but not the inhibitory effects of AngII on stimulus-secretion coupling. The AngII type 1 receptor (AT1R) antagonist losartan blocked both inhibition and facilitation of secretion by AngII. The results of this study show that activation of multiple types of G-proteins and transduction pathways by a single neuromodulator acting through one receptor type can produce concentration-dependent, bi-directional regulation of exocytosis.
Asunto(s)
Angiotensina II/farmacología , Señalización del Calcio/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Proteínas de Unión al GTP/metabolismo , Médula Suprarrenal/citología , Médula Suprarrenal/fisiología , Animales , Cafeína/farmacología , Calcio/metabolismo , Bovinos , Células Cultivadas , Células Cromafines/citología , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Femenino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Toxina del Pertussis , Proteína Quinasa C/antagonistas & inhibidores , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/fisiología , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We have used combined membrane capacitance measurements (C(m)) and voltage-clamp recordings to examine the mechanisms underlying modulation of stimulus-secretion coupling by a G(i/o)-coupled purinoceptor (P2Y) in adrenal chromaffin cells. P2Y purinoceptors respond to extracellular ATP and are thought to provide an important inhibitory feedback regulation of catecholamine release from central and sympathetic neurons. Inhibition of neurosecretion by other G(i/o)-protein-coupled receptors may occur by either inhibition of voltage-operated Ca(2+) channels or modulation of the exocytotic machinery itself. In this study, we show that the P2Y purinoceptor agonist 2-methylthio ATP (2-MeSATP) significantly inhibits Ca(2+) entry and changes in C(m) evoked by single 200 msec depolarizations or a train of 20 msec depolarizations (2.5 Hz). We found that P2Y modulation of secretion declines during a train such that only approximately 50% of the modulatory effect remains at the end of a train. The inhibition of both Ca(2+) entry and DeltaC(m) are also attenuated by large depolarizing prepulses and treatment with pertussis toxin. Inhibition of N-type, and to lesser extent P/Q-type, Ca(2+) channels contribute to the modulation of exocytosis by 2-MeSATP. The Ca(2+)-dependence of exocytosis triggered by either single pulses or trains of depolarizations was unaffected by 2-MeSATP. When Ca(2+) channels were bypassed and exocytosis was evoked by flash photolysis of caged Ca(2+), the inhibitory effect of 2-MeSATP was not observed. Collectively, these data suggest that inhibition of exocytosis by G(i/o)-coupled P2Y purinoceptors results from inhibition of Ca(2+) channels and the Ca(2+) signal controlling exocytosis rather than a direct effect on the secretory machinery.
Asunto(s)
Médula Suprarrenal/fisiología , Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Médula Suprarrenal/citología , Animales , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Exocitosis/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores Purinérgicos P2/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
1 Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. 2 Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG-encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4+/-0.4 microM (mean+/-s.e.mean) and a Hill coefficient of 1.17+/-0.03 (n = 5). 3 microM amitriptyline inhibited IHERG by 34+/-6% (n = 3). The inhibition showed only weak voltage dependence. 3 Using an 'envelope of tails' comprised of pulses to +20 mV of varying durations, the tau of activation was found to be 155+/-30 ms for control and 132+/-26 ms for 3 microM imipramine (n = 5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine-mediated HERG channel inhibition. 4 Taking current measurements every second during a 10 s depolarizing pulse from -80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). 5 A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at -80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The tau of inactivation was 8+/-2 ms and 5.6+/-0.4 ms (n = 5) in the absence and presence of 3 microM imipramine, respectively, and currents inactivated to a similar extent. 6 Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.
Asunto(s)
Amitriptilina/farmacología , Antidepresivos Tricíclicos/farmacología , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Imipramina/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Transactivadores , Animales , Células CHO , Cricetinae , ADN/biosíntesis , Canal de Potasio ERG1 , Estimulación Eléctrica , Canales de Potasio Éter-A-Go-Go , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio/biosíntesis , Regulador Transcripcional ERGRESUMEN
Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.
Asunto(s)
Calcio/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas de la Membrana , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Calcio/análisis , Línea Celular , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Células PC12 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Proteínas Tirosina Fosfatasas Clase 8 Similares a ReceptoresRESUMEN
1. Oxytocin and vasopressin secretion from the neurohypophysis (NHP) is evoked by strongly patterned bursts of action potentials. We studied excitation-secretion coupling in single isolated terminals of rat NHP using patch clamp and capacitance detection techniques. 2. The secretory response evoked by trains of depolarizing pulses consisted of two discrete phases. Ca2+ entry during pulses early in the train did not elicit secretion. Exocytotic responses began only after a characteristic amount of total Ca2+ entry called "threshold". 3. In the postthreshold secretory phase, exocytotic events occurred during or immediately after depolarizing pulses, indicating that the final Ca(2+)-dependent step is triggered by high Ca2+ concentrations near the plasma membrane that dissipate rapidly after channel closure. Secretion was sensitive to both the concentration and species of Ca2+ chelator. BAPTA, a Ca2+ chelator with rapid Ca2+ binding kinetics, was more effective than EGTA in diminishing secretion. 4. The "threshold" amount of Ca2+ was determined by the concentration, but not species, of Ca2+ chelator. The threshold value was constant even when Ca2+ entry parameters were varied over a broad range of current amplitudes, pulse durations, and number of pulses, indicating that it did not require high Ca2+ concentrations near the plasma membrane. 5. These results suggest that the secretory response to a train of pulses consists of a Ca(2+)-dependent preparatory step that must be completed before subsequent Ca2+ entry can elicit exocytosis. 6. Exocytotic responses during single trains showed strong depression at a step subsequent to Ca2+ entry. Recovery from depression required 30-60 sec. 7. The properties of threshold secretion observed in NHP terminals are discussed in terms of current models of secretion.
Asunto(s)
Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/fisiología , Neurohipófisis/metabolismo , Animales , Bario/farmacología , Canales de Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Conductividad Eléctrica , Potenciales Evocados/efectos de los fármacos , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Sistemas Neurosecretores/fisiología , Técnicas de Placa-Clamp , Neurohipófisis/citología , Neurohipófisis/efectos de los fármacos , RatasRESUMEN
ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X1 (or P2X3) receptor had strong desensitization, whereas currents in cells expressing P2X2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X1 receptor of either one of two 34-residue segments from the P2X2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X2 receptor only by providing both these segments of the P2X1 (or P2X3) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.
Asunto(s)
Adenosina Trifosfato/farmacología , Canales Iónicos/fisiología , Receptores Purinérgicos/fisiología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/fisiología , Cartilla de ADN , Potenciales Evocados/efectos de los fármacos , Femenino , Canales Iónicos/biosíntesis , Canales Iónicos/química , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/fisiología , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos/biosíntesis , Receptores Purinérgicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , XenopusRESUMEN
An opioid-like receptor has been cloned by several groups of researchers and recently shown to be activated by an endogenous heptadecapeptide termed orphanin FQ (or nociceptin). We isolated the corresponding mouse cDNA and coexpressed it in Xenopus laevis oocytes with the potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (CIR, rcKATP). Orphanin FQ evoked potassium currents, with 50% of the maximal effect at approximately 1 nM; [Tyr1]orphanin FQ was equally effective, and des-pheorphanin FQ was without activity. Dynorphin A, dynorphin(1-9), dynorphin(1-13), and alpha-neoendorphin were > 100 times less potent, and other agonists active at mu-, delta-, and kappa-opioid receptors had no effect. Naloxone (1 microM) and norbinaltorphimine (1 microM) had no antagonist action. Conversely, oocytes expressing kappa receptors responded to dynorphin (half-maximal concentration, 0.3 nM) but not to orphanin FQ. Thus, both kappa and orphanin FQ receptors readily couple to potassium channels, but the highly selective activation by dynorphin and orphanin FQ is consistent with distinct functional pathways in vivo.
Asunto(s)
Oocitos/fisiología , Péptidos Opioides/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/biosíntesis , Receptores Opioides kappa/metabolismo , Receptores Opioides/biosíntesis , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Clonación Molecular , Dinorfinas/farmacología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Expresión Génica , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Antagonistas de Narcóticos , Oligopéptidos/farmacología , Oocitos/efectos de los fármacos , Péptidos Opioides/farmacología , Fragmentos de Péptidos/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Ratas , Receptores Opioides/aislamiento & purificación , Receptores Opioides/metabolismo , Receptores Opioides kappa/biosíntesis , Especificidad por Sustrato , Xenopus laevis , Receptor de Nociceptina , NociceptinaRESUMEN
Xenopus oocytes were injected with RNAs for the two inward-rectifier potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (rcKATP or CIR) in addition to RNA from the neuroblastoma cell line KAN-TS. Potassium currents were evoked by neuropeptide Y in oocytes injected with polyadenylated RNA or with cRNA from pools of a neuroblastoma (KAN-TS) cDNA library, and progressive subdivision of responding pools yielded a single cDNA. The encoded protein contains 381 amino acids, has the seven hydrophobic domains characteristic of G protein-coupled receptors, and is 31% identical to the Y1 receptor: potassium currents were induced by neuropeptide Y (EC50=60pm) and Y2-selective analogues. Coexpression with potassium channel subunits will be a generally useful method for the cloning of G protein-coupled receptors.
Asunto(s)
Neuropéptido Y/metabolismo , Canales de Potasio/genética , Receptores de Neuropéptido Y/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Neuroblastoma , Neuropéptido Y/farmacología , Oocitos/fisiología , Oocitos/ultraestructura , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , ARN/genética , ARN Mensajero/genética , Receptores de Neuropéptido Y/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , XenopusRESUMEN
The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.
Asunto(s)
Bario/farmacología , Sistema Cromafín/efectos de los fármacos , Exocitosis/fisiología , Neurohipófisis/efectos de los fármacos , Animales , Calcio/farmacología , Cationes/farmacología , Bovinos , Cinética , Factores de TiempoRESUMEN
Stimulus-secretion coupling in bovine chromaffin cells was investigated with whole-cell patch-clamp recordings and capacitance detection techniques to monitor exocytosis in response to trains of depolarizing pulses. Two kinetically discrete modes of exocytotic responses were observed. In one mode, the first depolarization of a train elicited a large increase in membrane capacitance (Cm; mean approximately 70 fF). This secretory mode was characterized by small Ca2+ requirements, relative insensitivity to the pipette Ca2+ chelator concentration, and rapid depletion of the secretory response. This mode of stimulus-secretion coupling was labile and was seen only in response to the first and, occasionally, the second stimulus train of whole-cell recordings. The second type of exocytotic response persisted for the remainder of the whole-cell recordings and consisted of two distinct phases. During the earliest pulses of a stimulus train, Ca2+ entry did not evoke Cm increases. Instead, Cm responses were elicited by later pulses, despite diminished Ca2+ entry per pulse caused by Ca2+ channel inactivation. The secretory phase was initiated after a specific "threshold" amount of Ca2+ had entered the cell, which was determined by the concentration, but not the binding kinetics, of the Ca2+ chelator in the pipette. In both the early and the secretory phases, the response of the cell was proportional to cumulative Ca2+ entry, regardless of current amplitude, pulse duration, or number of pulses. Threshold-type secretory kinetics has been described previously in peptide-secreting neurohypophysial (NHP) nerve terminals (Seward et al., 1995). Secretory kinetics with minimal Ca2+ requirements has not been observed in that preparation. Chromaffin cells appear to possess a broader repertoire of stimulus-secretion coupling modes than NHP terminals.
Asunto(s)
Potenciales de Acción/fisiología , Calcio/farmacología , Sistema Cromafín/fisiología , Exocitosis/fisiología , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Cinética , Técnicas de Placa-ClampRESUMEN
The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio/fisiología , Calcio/metabolismo , Exocitosis , Terminaciones Nerviosas/fisiología , Neurohipófisis/fisiología , Animales , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica , Activación del Canal Iónico , Cinética , Terminaciones Nerviosas/efectos de los fármacos , Ratas , Factores de TiempoRESUMEN
The traditional function of neurotransmitter-gated ion channels is to induce rapid changes in electrical activity. Channels that are Ca(2+)-permeable, such as N-methyl-D-aspartate receptors at depolarized membrane potentials, can have a broader repertoire of consequences, including changes in synaptic efficacy, developmental plasticity, and excitotoxicity. Neuronal nicotinic receptors for acetylcholine (nAChRs) are usually less Ca(2+)-permeable than N-methyl-D-aspartate receptors but have a significant Ca2+ permeability, which is greater at negative potentials. Here we report that in neuroendocrine cells, activation of nAChRs can trigger exocytosis at hyperpolarized potentials. We used whole-cell patch-clamp recordings to record currents and the capacitance detection technique to monitor exocytosis in isolated bovine chromaffin cells. Stimulation of nAChRs at hyperpolarized potentials (-60 or -90 mV) evokes a large current and a maximal capacitance increase corresponding to the fusion of approximately 200 large dense-core vesicles. The amount of exocytosis is controlled both by the Ca2+ influx through nAChRs and by a contribution from thapsigargin-sensitive Ca2+ sequestering stores. This is a form of neurotransmitter action in which activation of nAChRs triggers secretion through an additional coupling pathway that coexists with classical voltage-dependent Ca2+ entry.
Asunto(s)
Médula Suprarrenal/fisiología , Calcio/metabolismo , Exocitosis , Nicotina/farmacología , Receptores Nicotínicos/fisiología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Bovinos , Células Cultivadas , Citosol/metabolismo , Potenciales Evocados/efectos de los fármacos , Exocitosis/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Terpenos/farmacología , Tapsigargina , Factores de Tiempo , Tubocurarina/farmacologíaRESUMEN
Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.
Asunto(s)
Actinas/metabolismo , Corteza Suprarrenal/fisiología , Gránulos Cromafines/ultraestructura , Exocitosis , Actinas/efectos de los fármacos , Actinas/ultraestructura , Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/ultraestructura , Animales , Bovinos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/fisiología , Electrofisiología/métodos , Epinefrina/metabolismo , Cinética , Potenciales de la Membrana , Microscopía Electrónica , Nicotina/farmacología , Norepinefrina/metabolismo , Potasio/farmacología , Acetato de Tetradecanoilforbol/farmacología , Grabación en VideoRESUMEN
Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (-30 to -20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from -90 to +10 mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10-300 microM) and gadolinium (10-30 microM) as well as by high concentrations of nickel and cobalt. omega Conus toxin (1 microM) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3-10 microM) and antagonists, nifedipine (3-10 microM) and nimodipine (10 microM) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.