RESUMEN
Small GTPase cycled between the GDP-bound inactive state and GTP-bound active state, catalyzed by guanine nucleotide exchange factors (GEFs). Guanine nucleotide exchange assay was a direct way to investigate the specificity, activity, and kinetics of GEFs. The N-methylanthraniloyl derivative of GDP (mantGDP), which was bound to small GTPase, served as a substitution for labeled small GTPase involved in bioluminescent, colorimetric, or radioactive methods due to its safety and sensitivity. In this study, we present an economical and efficient approach to prepare qualified mantGDP-bound CDC42, a member of the Rho GTPase family. In our protocol, with a Kd value of 0.048 µM, alkaline phosphatase hydrolysis of CDC42 increased mantGDP binding affinity to CDC42, allowing mant-nucleotide associating onto CDC42 more easily. Only 1.5-fold molar excess of mantGDP was required to prepare mantGDP-bound CDC42 without nonhydrolyzable GTP analog and high performance liquid chromatography. The mantGDP-bound CDC42 was verified to be efficient for measuring the guanine nucleotide exchange activity of VAV2.
Asunto(s)
Pruebas de Enzimas/métodos , Guanosina Difosfato/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Fosfatasa Alcalina/metabolismo , Calorimetría , Guanosina Difosfato/análogos & derivados , Humanos , Hidrólisis , Cinética , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/genéticaRESUMEN
This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m7 GMP), 7-methylguanosine 5'-O-diphosphate (m7 GDP), 7-methyl-2'-deoxyguanosine 5'-O-triphosphate (m7 2'dGTP), and 7-methylguanosine 5'-O-triphosphate (m7 GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7-methylguanosine nucleotides.
Asunto(s)
Guanosina Difosfato/análogos & derivados , Análogos de Caperuza de ARN/síntesis química , Guanosina Difosfato/síntesis química , Guanosina Difosfato/química , Indicadores y Reactivos/química , Metilación , Análogos de Caperuza de ARN/química , Solventes/química , Ésteres del Ácido Sulfúrico/químicaRESUMEN
Dopamine (D2) receptors provide autoinhibitory feedback onto dopamine neurons through well-known interactions with voltage-gated calcium channels and G protein-coupled inwardly-rectifying potassium (GIRK) channels. Here, we reveal a third major effector involved in D2R modulation of dopaminergic neurons - the sodium leak channel, NALCN. We found that activation of D2 receptors robustly inhibits isolated sodium leak currents in wild-type mice but not in NALCN conditional knockout mice. Intracellular GDP-ßS abolished the inhibition, indicating a G protein-dependent signaling mechanism. The application of dopamine reliably slowed pacemaking even when GIRK channels were pharmacologically blocked. Furthermore, while spontaneous activity was observed in nearly all dopaminergic neurons in wild-type mice, neurons from NALCN knockouts were mainly silent. Both observations demonstrate the critical importance of NALCN for pacemaking in dopaminergic neurons. Finally, we show that GABA-B receptor activation also produces inhibition of NALCN-mediated currents. Therefore, we identify NALCN as a core effector of inhibitory G protein-coupled receptors.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Neuronas Dopaminérgicas/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Canales Iónicos/genética , Proteínas del Tejido Nervioso/genética , Receptores de Dopamina D2/metabolismo , Receptores de GABA-B/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Canales de Calcio Tipo N/genética , Dopamina/farmacología , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Expresión Génica , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Canales Iónicos/deficiencia , Transporte Iónico/efectos de los fármacos , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtomía , Proteínas del Tejido Nervioso/deficiencia , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Receptores de Dopamina D2/genética , Receptores de GABA-B/genética , Tionucleótidos/farmacología , Técnicas de Cultivo de Tejidos , Valina/análogos & derivados , Valina/farmacología , Área Tegmental Ventral/citología , Área Tegmental Ventral/metabolismoRESUMEN
BACKGROUND: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of Gi protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin. PURPOSE: The aim of the present work was to evaluate the potential of a Gi-protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages. MATERIALS AND METHODS: Guanosine 5'-O-(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1ß, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated. RESULTS: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1ß, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS. CONCLUSION: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages.
Asunto(s)
Guanosina Difosfato/análogos & derivados , Inflamación/prevención & control , Liposomas/administración & dosificación , Monocitos/efectos de los fármacos , Tionucleótidos/farmacología , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Guanosina Difosfato/administración & dosificación , Guanosina Difosfato/farmacología , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Liposomas/química , Liposomas/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Tionucleótidos/administración & dosificación , Factor de Necrosis Tumoral alfaRESUMEN
Excitatory synaptic transmission in central synapses is modulated by serotonin (5-HT). The anterior cingulate cortex (ACC) is an important cortical region for pain perception and emotion. ACC neurons receive innervation of projecting serotonergic nerve terminals from raphe nuclei, but the possible effect of 5-HT on excitatory transmission in the ACC has not been investigated. In the present study, we investigated the role of 5-HT on glutamate neurotransmission in the ACC slices of adult mice. Bath application of 5-HT produced dose-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs). Paired pulse ratio (PPR) was significantly increased, indicating possible presynaptic effects of 5-HT. Consistently, bath application of 5-HT significantly decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. After postsynaptic application of G protein inhibitor GDP-ß-S, 5-HT produced inhibition of eEPSCs was significantly reduced. Finally, NAN-190, an antagonist of 5-HT1A receptor, significantly reduced postsynaptic inhibition of 5-HT and abolished presynaptic inhibition. Our results strongly suggest that presynaptic as well as postsynaptic 5-HT receptor including 5-HT1A subtype receptor may contribute to inhibitory modulation of glutamate release as well as postsynaptic responses in the ACC.
Asunto(s)
Giro del Cíngulo/fisiología , Serotonina/farmacología , Transmisión Sináptica/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Giro del Cíngulo/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Piperazinas/farmacología , Receptor de Serotonina 5-HT1A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRasG12C using GDP equipped with an electrophilic group at the ß-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such ß-modified derivatives for Ras of at least 104 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.
Asunto(s)
Guanosina Trifosfato/metabolismo , Nucleótidos/metabolismo , Nucleótidos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacología , Guanosina Trifosfato/química , Cinética , Modelos Biológicos , Estructura Molecular , Nucleótidos/química , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes , Proteínas Son Of Sevenless/química , Proteínas Son Of Sevenless/metabolismo , Relación Estructura-ActividadRESUMEN
Elimination of the 5' cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5'-3' exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1-7 complex, Pat1, Edc1-Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1-Dcp2 enzyme bound to its product (m7GDP) and its potent activator Edc3. This structure of the Dcp1-Dcp2 complex bound to a cap analog further explains previously published data on substrate binding and provides hints as to the mechanism of Edc3-mediated Dcp2 activation.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Guanosina Difosfato/análogos & derivados , Saccharomycetales/metabolismo , Cristalografía por Rayos X , Endorribonucleasas/química , Activación Enzimática , Proteínas Fúngicas/química , Guanosina Difosfato/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad del ARN , Saccharomycetales/químicaRESUMEN
The dorsolateral striatum mediates habit formation, which is expedited by exposure to alcohol. Across species, alcohol exposure disinhibits the DLS by dampening GABAergic transmission onto this structure's principal medium spiny projection neurons (MSNs), providing a potential mechanistic basis for habitual alcohol drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. To examine this, we used a combination of whole-cell patch-clamp recordings and optogenetics to demonstrate that ethanol potently depresses both MSN- and fast-spiking interneuron (FSI)-MSN GABAergic synaptic transmission in the DLS. Concentrating on the powerfully inhibitory FSI-MSN synapse, we further show that acute exposure of ethanol (50 mM) to striatal slices activates delta opioid receptors that reside on FSI axon terminals and negatively couple to adenylyl cyclase to induce a long-term depression of GABA release onto both direct and indirect pathway MSNs. These findings elucidate a mechanism through which ethanol may globally disinhibit the DLS.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Cuerpo Estriado/citología , Etanol/farmacología , Neuronas/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Receptores Opioides delta/metabolismo , Analgésicos Opioides/farmacología , Animales , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antagonistas de Narcóticos/farmacología , Parvalbúminas/genética , Parvalbúminas/metabolismo , Terminales Presinápticos/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores Opioides delta/antagonistas & inhibidores , Tionucleótidos/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Glycine receptors (GlyRs) permeable to chloride only mediate tonic inhibition in the cerebral cortex where glycinergic projection is completely absent. The functional modulation of GlyRs was largely studied in subcortical brain regions with glycinergic transmissions, but the function of cortical GlyRs was rarely addressed. Serotonin could broadly modulate many ion channels through activating 5-HT2 receptor, but whether cortical GlyRs are subjected to serotonergic modulation remains unexplored. The present study adopted patch clamp recordings to examine functional regulation of strychnine-sensitive GlyRs currents in cultured cortical neurons by DOI (2,5-Dimethoxy-4-iodoamphetamine), a 5-HT2A/C receptor agonist. DOI caused a concentration-dependent reduction of GlyR currents with unchanged reversal potential. This reduction was blocked by the selective receptor antagonists (ritanserin and risperidone) and G protein inhibitor (GDP-ß-s) demonstrated that the reducing effect of DOI on GlyR current required the activation of 5-HT2A/C receptors. Strychnine-sensitive tonic currents revealed the inhibitory tone mediated by nonsynaptic GlyRs, and DOI similarly reduced the tonic inhibition. The impaired microtube-dependent trafficking or clustering of GlyRs was thought to be involved in that nocodazole as a microtube depolymerizing drug largely blocked the inhibition mediated by 5-HT2A/C receptors. Our results suggested that activation of 5-HT2A/C receptors might suppress cortical tonic inhibition mediated by GlyRs, and the findings would provide important insight into serotonergic modulation of tonic inhibition mediated by GlyRs, and possibly facilitate to develop the therapeutic treatment of neurological diseases such as tinnitus through regulating cortical GlyRs.
Asunto(s)
Corteza Auditiva/metabolismo , Neuronas/metabolismo , Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2C/metabolismo , Receptores de Glicina/metabolismo , Anfetaminas/farmacología , Animales , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Canales Iónicos/metabolismo , Microtúbulos/metabolismo , Nocodazol/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Risperidona/farmacología , Ritanserina/farmacología , Agonistas del Receptor de Serotonina 5-HT2/farmacología , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Estricnina/farmacología , Tionucleótidos/farmacología , Moduladores de Tubulina/farmacologíaRESUMEN
Directly inhibiting oncogenic RAS proteins has proven to be an arduous task, as after more than thirty years of intensive investigation, no clinically relevant therapies exist. Recently, two classes of selective small molecule inhibitors that target a cysteine-containing RAS mutant have been developed, representing the first directed approaches to specifically inhibit an oncogenic KRAS mutant. In this mini-review, we first assess the development and targeting strategies associated with novel cysteine-directed RAS inhibitors. Next, we describe the variable oncogenic potency of the KRAS G12C mutant when compared to other KRAS G12 mutants. Lastly, we evaluate how the redox properties of KRAS G12C may play a role in differential signaling and tumorigenic potency of the oncogene, the efficacy of small molecules targeting this specific RAS mutant and further development of directed oncogenic RAS inhibitors.
Asunto(s)
Proteínas ras/metabolismo , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Cisteína/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Guanosina Difosfato/uso terapéutico , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Proteínas ras/antagonistas & inhibidores , Proteínas ras/genéticaRESUMEN
We previously reported that a Pt(IV) complex, [Pt(IV)(dach)Cl4] [trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum(IV)] binds to the N7 of 5'-dGMP (deoxyguanosine-5'-monophosphate) at a relatively fast rate and oxidizes it to 8-oxo-5'-dGMP. Here, we further studied the kinetics of the oxidation of 5'-dGMP by the Pt(IV) complex. The electron transfer rate constants between 5'-dGMP and Pt(IV) in [H8-5'-dGMP-Pt(IV)] and [D8-5'-dGMP-Pt(IV)] were similar, giving a small value of the kinetic isotope effect (KIE: 1.2 ± 0.2). This small KIE indicates that the deprotonation of H8 in [H8-5'-dGMP-Pt(IV)] is not involved in the rate-determining step in the electron transfer between guanine (G) and Pt(IV). We also studied the reaction of 5'-dGDP (deoxyguanosine-5'-diphosphate) and 5'-dGTP (deoxyguanosine-5'-triphosphate) with the Pt(IV) complex. Our results showed that [Pt(IV)(dach)Cl4] oxidized 5'-dGDP and 5'-dGTP to 8-oxo-5'-dGDP and 8-oxo-5'-dGTP, respectively, by the same mechanism and kinetics as for 5'-dGMP. The Pt(IV) complex binds to N7 followed by a two-electron inner sphere electron transfer from G to Pt(IV). The reaction was catalyzed by Pt(II) and occurred faster at higher pH. The electron transfer was initiated by either an intramolecular nucleophilic attack by any of the phosphate groups or an intermolecular nucleophilic attack by free OH(-) in the solution. The rates of reactions for the three nucleotides followed the order: 5'-dGMP > 5'-dGDP > 5'-dGTP, indicating that the bulkier the phosphate groups are, the slower the reaction is, due to the larger steric hindrance and rotational barrier of the phosphate groups.
Asunto(s)
Complejos de Coordinación/química , Nucleótidos de Guanina/metabolismo , Platino (Metal)/metabolismo , Complejos de Coordinación/metabolismo , Nucleótidos de Desoxiguanina/química , Nucleótidos de Desoxiguanina/metabolismo , Nucleótidos de Guanina/química , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Oxidación-Reducción , Platino (Metal)/química , Factores de TiempoRESUMEN
Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK.
Asunto(s)
Colecistoquinina/metabolismo , Núcleo Hipotalámico Dorsomedial/citología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Colecistoquinina/farmacología , GABAérgicos/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Técnicas In Vitro , Masculino , Técnicas de Placa-Clamp , Péptidos/farmacología , Proglumida/análogos & derivados , Proglumida/farmacología , Quinazolinonas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/antagonistas & inhibidores , Proteína 25 Asociada a Sinaptosomas/metabolismo , Tionucleótidos/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the µ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-ß-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.
Asunto(s)
Baclofeno/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo T/metabolismo , Agonistas de Receptores GABA-B/farmacología , Receptores de GABA-B/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Ditiotreitol/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Ganglios Espinales , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Células HEK293 , Humanos , Nocicepción/fisiología , Dolor/metabolismo , Dolor/fisiopatología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Tionucleótidos/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoosan (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The wholecell patchclamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura2acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4diphenylacetoxyNmethylpiperidine methiodide muscarinic M3 receptor antagonist inhibited DSinduced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'[ßthio] diphosphate (GDPßS; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DSinduced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DSinduced pacemaker depolarizations. Pretreatment with Ca2+free solution and thapsigargin, a Ca2+ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DSinduced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G proteindependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.
Asunto(s)
Células Intersticiales de Cajal/efectos de los fármacos , Dolor/tratamiento farmacológico , Fitoterapia , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Diaminas/farmacología , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Piperidinas/farmacología , Plantas Medicinales/efectos adversos , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/metabolismo , Tapsigargina/farmacología , Tionucleótidos/metabolismoRESUMEN
CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel ß-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-ß-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Señalización del Calcio/efectos de los fármacos , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Activación Enzimática/efectos de los fármacos , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Cinética , Técnicas de Placa-Clamp , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tionucleótidos/metabolismoRESUMEN
It has been reported that kappa opioid receptor (KOR) is expressed in the paraventricular nucleus of thalamus (PVT), a brain region associated with arousal, drug reward and stress. Although intra-PVT infusion of KOR agonist was found to inhibit drug-seeking behavior, it is still unclear whether endogenous KOR agonists directly regulate PVT neuron activity. Here, we investigated the effect of the endogenous KOR agonist dynorphin-A (Dyn-A) on the excitability of mouse PVT neurons at different developmental ages. We found Dyn-A strongly inhibited PVT neurons through a direct postsynaptic hyperpolarization. Under voltage-clamp configuration, Dyn-A evoked an obvious outward current in majority of neurons tested in anterior PVT (aPVT) but only in minority of neurons in posterior PVT (pPVT). The Dyn-A current was abolished by KOR antagonist nor-BNI, Ba(2+) and non-hydrolyzable GDP analogue GDP-ß-s, indicating that Dyn-A activates KOR and opens G-protein-coupled inwardly rectifying potassium channels in PVT neurons. More interestingly, by comparing Dyn-A currents in aPVT neurons of mice at various ages, we found Dyn-A evoked significant larger current in aPVT neurons from mice around prepuberty and early puberty stage. In addition, KOR activation by Dyn-A didn't produce obvious desensitization, while mu opioid receptor (MOR) activation induced obvious desensitization of mu receptor itself and also heterologous desensitization of KOR in PVT neurons. Together, our findings indicate that Dyn-A activates KOR and inhibits aPVT neurons in mice at various ages especially around puberty, suggesting a possible role of KOR in regulating aPVT-related brain function including stress response and drug-seeking behavior during adolescence.
Asunto(s)
Dinorfinas/metabolismo , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Receptores Opioides kappa/metabolismo , Animales , Bario/administración & dosificación , Cationes/administración & dosificación , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Guanosina Difosfato/administración & dosificación , Guanosina Difosfato/análogos & derivados , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BL , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Neuronas/efectos de los fármacos , Neurotransmisores/administración & dosificación , Neurotransmisores/metabolismo , Núcleo Hipotalámico Paraventricular/crecimiento & desarrollo , Técnicas de Placa-Clamp , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inhibidores , Maduración Sexual/fisiología , Tionucleótidos/administración & dosificación , Técnicas de Cultivo de TejidosRESUMEN
Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-ß2/Transportin (Kapß2). SiRNA-directed Kapß2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.
Asunto(s)
Neoplasias del Sistema Nervioso Central/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glioblastoma/metabolismo , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Citosol/metabolismo , Ensayo de Inmunoadsorción Enzimática , Regulación Neoplásica de la Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Humanos , Peso Molecular , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Tionucleótidos/metabolismo , TransfecciónRESUMEN
Although involved in various physiological functions, nucleoside bis-phosphate analogues and their metal-ion complexes have been scarcely studied. Hence, here, we explored the solution conformation of 2'-deoxyadenosine- and 2'-deoxyguanosine-3',5'-bisphosphates, 3 and 4, d(pNp), as well as their Zn(2+)/Mg(2+) binding sites and binding-modes (i.e. inner- vs. outer-sphere coordination), acidity constants, stability constants of their Zn(2+)/Mg(2+) complexes, and their species distribution. Analogues 3 and 4, in solution, adopted a predominant Southern ribose conformer (ca. 84%), gg conformation around C4'-C5' and C5'-O5' bonds, and glycosidic angle in the anti-region (213-270°). (1)H- and (31)P-NMR experiments indicated that Zn(2+)/Mg(2+) ions coordinated to P5' and P3' groups of 3 and 4 but not to N7 nitrogen atom. Analogues 3 and 4 formed ca. 100-fold more stable complexes with Zn(2+)vs. Mg(2+)-ions. Complexes of 3 and 4 with Mg(2+) at physiological pH were formed in minute amounts (11% and 8%, respectively) vs. Zn(2+) complexes (46% and 44%). Stability constants of Zn(2+)/Mg(2+) complexes of analogues 3 and 4 (log KML(M) = 4.65-4.75/2.63-2.79, respectively) were similar to those of the corresponding complexes of ADP and GDP (log KML(M) = 4.72-5.10/2.95-3.16, respectively). Based on the above findings, we hypothesized that the unexpectedly low log K values of Zn(2+)-d(pNp) as compared to Zn(2+)-NDP complexes, are possibly due to formation of outer-sphere coordination in the Zn(2+)-d(pNp) complex vs. inner-sphere in the NDP-Zn(2+) complex, in addition to loss of chelation to N7 nitrogen atom in Zn(2+)-d(pNp). Indeed, explicit solvent molecular dynamics simulations of 1 and 3 for 100 ns supported this hypothesis.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Quelantes/química , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/química , Zinc/química , Sitios de Unión , Complejos de Coordinación/química , Técnicas Electroquímicas , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Iones/química , Cinética , Magnesio/química , Espectroscopía de Resonancia Magnética , Conformación Molecular , Simulación de Dinámica MolecularRESUMEN
By activating their receptors (OX1R and OX2R) orexin-A/B regulate wake/sleeping states, feeding behaviors, but the function of these peptides in the retina remains unknown. Using patch-clamp recordings and calcium imaging in rat isolated retinal cells, we demonstrated that orexin-A suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-preferring receptor-mediated currents (AMPA-preferring currents) in ganglion cells (GCs) through OX1R, but potentiated those in amacrine cells (ACs) through OX2R. Consistently, in rat retinal slices orexin-A suppressed light-evoked AMPA-preferring receptor-mediated excitatory postsynaptic currents in GCs, but potentiated those in ACs. Intracellular dialysis of GDP-ß-S or preincubation with the Gi/o inhibitor pertussis toxin (PTX) abolished both the effects. Either cAMP/the protein kinase A (PKA) inhibitor Rp-cAMP or cGMP/the PKG blocker KT5823 failed to alter the orexin-A effects. Whilst both of them involved activation of protein kinase C (PKC), the effects on GCs and ACs were respectively eliminated by the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor and phosphatidylcholine (PC)-PLC inhibitor. Moreover, in GCs orexin-A increased [Ca(2+)]i and the orexin-A effect was blocked by intracellular Ca(2+)-free solution and by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. In contrast, orexin-A did not change [Ca(2+)]i in ACs and the orexin-A effect remained in intracellular or extracellular Ca(2+)-free solution. We conclude that a distinct Gi/o/PI-PLC/IP3/Ca(2+)-dependent PKC signaling pathway, following the activation of OX1R, is likely responsible for the orexin-A effect on GCs, whereas a Gi/o/PC-PLC/Ca(2+)-independent PKC signaling pathway, following the activation of OX2R, mediates the orexin-A effect on ACs. These two actions of orexin-A, while working in concert, provide a characteristic way for modulating information processing in the inner retina.
Asunto(s)
Células Amacrinas/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Orexinas/farmacología , Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Células Amacrinas/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Colorantes Fluorescentes/metabolismo , Ácido Glutámico/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Antagonistas de los Receptores de Orexina/farmacología , Técnicas de Placa-Clamp , Ratas , Células Ganglionares de la Retina/metabolismo , Rianodina/farmacología , Transducción de Señal/efectos de los fármacos , Tionucleótidos/farmacologíaRESUMEN
Eukaryotic 5' mRNA cap structures participate to the post-transcriptional control of gene expression before being released by the two main mRNA decay pathways. In the 3'-5' pathway, the exosome generates free cap dinucleotides (m7GpppN) or capped oligoribonucleotides that are hydrolyzed by the Scavenger Decapping Enzyme (DcpS) forming m7GMP. In the 5'-3' pathway, the decapping enzyme Dcp2 generates m7GDP. We investigated the fate of m7GDP and m7GpppN produced by RNA decay in extracts and cells. This defined a pathway involving DcpS, NTPs and the nucleoside diphosphate kinase for m7GDP elimination. Interestingly, we identified and characterized in vitro and in vivo a new scavenger decapping enzyme involved in m7GpppN degradation. We show that activities mediating cap elimination identified in yeast are essentially conserved in human. Their alteration may contribute to pathologies, possibly through the interference of cap (di)nucleotide with cellular function.