RESUMO
The P2X5 receptor, an ATP-gated cation channel, is believed to be involved in tumor development, inflammatory bone loss and inflammasome activation after bacterial infection. Therefore, it is a worthwhile pharmacological target to treat the corresponding diseases, especially in minority populations that have a gene variant coding for functional homotrimeric P2X5 channels. Here, we investigated the effects of dihydropyridines on the human full-length P2X5 receptor (hP2X5FL) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage clamp method. Agonist dependency, kinetics and permeation behavior, including Cl- permeability, were similar to hP2X5FL expressed in HEK293 or 1321N1 cells. Additionally, 1,4-dihydropyridines have been shown to interact with various other purinergic receptors, and we have examined them as potential hP2X5 modulators. Of seven commercially available and four newly synthesized dihydropyridines tested at hP2X5FL, only amlodipine exerted an inhibitory effect, but only at a high concentration of 300 µM. Isradipine and-even more-nimodipine stimulated ATP-induced currents in the low micromolar range. We conclude that common dihydropyridines or four new derivatives of amlodipine are not suitable as hP2X5 antagonists, but amlodipine might serve as a lead for future synthesis to increase its affinity. Furthermore, a side effect of nimodipine therapy could be a stimulatory effect on inflammatory processes.
Assuntos
Di-Hidropiridinas , Trifosfato de Adenosina/farmacologia , Di-Hidropiridinas/farmacologia , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Receptores PurinérgicosRESUMO
The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327-357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4.
Assuntos
Ativação do Canal Iônico , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Carbocianinas/química , Cisteína/química , Cisteína/genética , Ligação de Hidrogênio , Ativação do Canal Iônico/genética , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Relação Estrutura-AtividadeRESUMO
Microglia cells represent the immune system of the central nervous system. They become activated by ATP released from damaged and inflamed tissue via purinergic receptors. Ionotropic purinergic P2X4 and P2X7 receptors have been shown to be involved in neurological inflammation and pain sensation. Whether the two receptors assemble exclusively as homotrimers or also as heterotrimers is still a matter of debate. We investigated the expression of P2X receptors in BV-2 microglia cells applying the whole-cell voltage-clamp technique. We dissected P2X4 and P2X7 receptor-mediated current components by using specific P2X4 and P2X7 receptor blockers and by their characteristic current kinetics. We found that P2X4 and P2X7 receptors are activated independently from each other, indicating that P2X4/P2X7 heteromers are not of functional significance in these cells. The pro-inflammatory mediators lipopolysaccharide and interferon γ, if applied in combination, upregulated P2X4, but not P2X7 receptor-dependent current components also arguing against phenotypically relevant heteromerization of P2X4 and P2X7 receptor subunits.
Assuntos
Microglia/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Células Cultivadas , Feminino , Interferon gama/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Xenopus laevis/metabolismoRESUMO
We report the activation of outwardly rectifying anion currents by sphingosine-1-phosphate (S1P) in the murine macrophage cell line RAW 264.7. The S1P-induced current is mainly carried by anions, because the reversal potential of the current was shifted by replacement of extracellular Cl(-) by glutamate(-) but not when extracellular Na(+) was substituted by Tris(+). The inhibition of the current by hypertonic extracellular or hypotonic intracellular solution as well as the inhibitory effects of NPPB, tamoxifen, and glibenclamide indicates that the anion current is mediated by volume-regulated anion channels (VRAC). The S1P effect was blocked by intracellular GDPßS and W123, which points to signaling via the S1P receptor 1 (S1PR1) and G proteins. As cytochalasin D diminished the action of S1P, we conclude that the actin cytoskeleton is involved in the stimulation of VRAC. S1P and hypotonic extracellular solution induced secretion of ATP from the macrophages, which in both cases was blocked in a similar way by typical VRAC blockers. We suppose that the S1P-induced ATP secretion in macrophages via activation of VRAC constitutes a functional link between sphingolipid and purinergic signaling in essential processes such as inflammation and migration of leukocytes as well as phagocytosis and the killing of intracellular bacteria.
Assuntos
Trifosfato de Adenosina/metabolismo , Tamanho Celular , Cloretos/metabolismo , Canais Iônicos/metabolismo , Lisofosfolipídeos/farmacologia , Macrófagos/metabolismo , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Citocalasina D/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Glibureto/farmacologia , Canais Iônicos/antagonistas & inibidores , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Nitrobenzoatos/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Sódio/metabolismo , Esfingosina/farmacologia , Tamoxifeno/farmacologiaRESUMO
The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum.
Assuntos
Sinalização do Cálcio , Canais de Cloreto/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Potenciais de Ação , Ambystoma mexicanum , Animais , Anoctamina-1 , Anoctaminas , Linhagem Celular Tumoral , Humanos , Camundongos , XenopusRESUMO
The neurohormone melatonin is synthesized by the pineal gland under the stimulation of norepinephrine (NE). Its synthesis is inhibited by acetylcholine (ACh) and by insulin. Type 2 diabetic Goto Kakizaki (GK) rats have higher insulin and lower melatonin plasma levels than healthy Wistar rats. We investigate membrane potentials and currents of isolated pinealocytes in both rat strains and the influence of NE, ACh and insulin by using the perforated patch whole cell clamp technique. Pinealocyte membranes displayed a high resting Na(+) conductance. Stimulation with NE further increased this Na(+) conductance, which led to a slight depolarization in unclamped cells. The amplitude of the NE-evoked current was similar in both rat strains but the current fraction carried by Na(+) was stronger in GK rats. Stimulation with ACh induced a transient inward current and depolarization. These effects were much more pronounced in the pinealocytes of GK rats. The NE-induced current, the ACh-induced current and the membrane depolarization were reduced by pre-administration of insulin in Wistar pinealocytes. Our results provide the first electrophysiological evidence for the modulation, by insulin, of the effects of NE and ACh in pinealocytes of normal rats. The pinealocytes of type 2 diabetic rats were not responsive to insulin. This might explain the reported correlation between the decreased insulin receptor mRNA transcript levels in GK rat pinealocytes and the lack of effect of insulin on ion channels in their cell membranes.
Assuntos
Acetilcolina/farmacologia , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Insulina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Norepinefrina/farmacologia , Glândula Pineal/patologia , Animais , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Masculino , Meglumina/farmacologia , Ratos , Ratos Wistar , Sódio/farmacologiaRESUMO
P2X receptors are trimeric adenosine-5'-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.
Assuntos
Trifosfato de Adenosina/química , Arginina/química , Ácido Glutâmico/química , Receptores Purinérgicos P2X2/química , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Feminino , Ativação do Canal Iônico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oócitos/fisiologia , Técnicas de Patch-Clamp , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/fisiologia , Receptores Purinérgicos P2X4/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática , Xenopus laevisRESUMO
Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR-1 and small LCL-HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman-Hodgkin-Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC4 (3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC4 (3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense.
Assuntos
Barbitúricos/química , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Isoxazóis/química , Potenciais da Membrana/efeitos dos fármacos , Algoritmos , Barbitúricos/metabolismo , Ionóforos de Cálcio/farmacologia , Calibragem , Linhagem Celular , Permeabilidade da Membrana Celular , Corantes Fluorescentes/metabolismo , Gramicidina/farmacologia , Humanos , Ionomicina/farmacologia , Isoxazóis/metabolismo , Modelos Biológicos , Coloração e Rotulagem/métodosRESUMO
TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
Assuntos
Canais de Cloreto/química , Animais , Anoctamina-1 , Reagentes de Ligações Cruzadas , Dimerização , Eletrofisiologia/métodos , Éxons , Células HEK293 , Humanos , Camundongos , Modelos Genéticos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Estrutura Quaternária de Proteína , Xenopus laevisRESUMO
P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.
Assuntos
Ânions , Receptores Purinérgicos P2X7/efeitos dos fármacos , Animais , Humanos , Mutação , Receptores Purinérgicos P2X7/genética , Xenopus laevisRESUMO
P2X7 receptors (P2X7Rs) are fast ATP4--gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP4- binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results.
Assuntos
Oócitos , Receptores Purinérgicos P2X7 , Trifosfato de Adenosina/metabolismo , Animais , Oócitos/metabolismo , RNA Complementar , Ratos , Receptores Purinérgicos P2X7/metabolismo , Xenopus laevis/metabolismoRESUMO
INTRODUCTION: In myotonic dystrophy, an increased potassium release upon ischemic forearm exercise has been previously described. However, it remains unclear whether this is specific for myotonic dystrophies or just due to distal muscular weakness. METHODS: Non-ischemic forearm test (NIFET) was performed and venous K+ concentration was measured at rest and at three different force levels (20-30%, 50-60%, 70-80%) related to maximal contraction force (MCF) in patients with distal myogenic (n = 7), neurogenic (n = 7) muscular weakness and healthy volunteers (n = 12). The specific K+ release was defined as K+ increase related to workload as force-time-integral during repetitive contraction. RESULTS: Workload was lower at all force levels in both disease groups compared to the control group. With increasing workload, the K+ concentrations increased in all study groups. Analysing individual force levels related to the maximum contraction force (MCF), a higher specific K+ release was measured at low force levels in myopathies (20-30% MCF) in comparison to higher force levels (p = 0.02). At 20-30% MCF, the specific K+ release was significantly higher in myogenic compared to neurogenic muscular weakness (p = 0.005). At 50-60% and 70-80% MCF, the specific K+ values converged and did not significantly differ between the three groups (p = 0.09 and p = 0.37). DISCUSSION: At low force levels, K+ efflux related to workload is higher in patients with myogenic in comparison to neurogenic distal paresis. Our results indicate a different regulation of K+ balance in neurogenic and myogenic muscular weakness possibly due to a different recruitment behaviour of motor units and the firing rate of motor neurons.
Assuntos
Doenças Musculares , Distrofia Miotônica , Exercício Físico , Humanos , Neurônios Motores , Debilidade Muscular/etiologia , Distrofia Miotônica/complicaçõesRESUMO
P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.
Assuntos
Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Imunofluorescência , Humanos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Patch clamp investigations of single ion channels give insight into the function of these proteins on the molecular level. Utilizing this technique, we performed detailed investigations of the human P2X7 receptor, which is a ligand gated ion channel opened by binding of ATP, like the other P2X receptor subtypes. P2X7 receptors become activated under pathological conditions of ATP release like hypoxia or cell destruction. They are involved in inflammatory and nociceptive reactions of the organism to these pathological events. Knowledge about the function of the P2X7 receptor might lead to a deeper insight into the signaling within these pathophysiological processes and to reveal targets of anti-inflammatory and anti-nociceptive therapies. We found that hP2X7 receptors become activated by ATP within a few milliseconds and are permeable only to cations. Their ion channel conductance remains constant across minutes of activation, which argues against dilation of the ion channel pore. Substitution of Na+ or Cl- ions not only influences the ion channel current amplitude but also the channel gating. Polar residues of the second transmembrane domains of the three protein subunits are important for ion conduction, with S342 constituting the ion selectivity filter and the gate of the channel. The specific long C-terminal domains are important for hP2X7 receptor ion channel function, as their loss strongly decreases ion channel currents.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X7/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Antagonistas Purinérgicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
Microglia cells are versatile players coordinating inflammatory and regenerative processes in the central nervous system in which sphingosine-1-phosphate (S1P)-mediated migration is essential. We investigated the involved signaling cascade by means of voltage clamp, measurement of ATP secretion, and wound healing assay in murine microglial BV-2 cells. S1P and extracellular hypoosmolar solution evoked an anion conductance of the cell membrane. The corresponding ion currents were inhibited by intracellular hypoosmolar solution and by the anion channel antagonists NPPB, tamoxifen, and carbenoxolone, pointing to the activation of volume-regulated anion channels (VRAC). The knockdown by siRNA indicates the involvement of LRRC8A subunits. The S1PR1-antagonist W123 and pertussis-toxin prevented the S1P-induced currents, showing the involvement of the Gi-protein-coupled S1P receptor 1 (S1PR1). Furthermore, S1P and hypoosmolar extracellular solution induced an increase of ATP levels in the supernatants of BV-2 cells, which was inhibited by NPPB, tamoxifen, and W123. S1P, ATP, and ADP stimulated cell migration into the scratch area. The inhibition of S1PR1 and the downstream Gi proteins hampered cell migration. Antagonists of VRAC were also able to diminish the migration of BV-2 cells. Furthermore, direct inhibition of ATP-gated P2X4 or P2X7 receptors or ADP-stimulated P2Y12 receptors blocked the stimulating effects of S1P on BV-2 cell migration. We conclude that there is an interaction between S1P receptors and purinergic receptors mediated by an S1P-induced ATP release via VRAC and that the amount of released ATP is capable of stimulating cell migration of BV-2 microglia cells via activation of P2X4, P2X7, and P2Y12 receptors.
Assuntos
Trifosfato de Adenosina/metabolismo , Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Microglia/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Células Cultivadas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Técnicas de Patch-Clamp , Esfingosina/farmacologia , TransfecçãoRESUMO
The EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important effector of the renin-angiotensin-aldosterone-system and elicits pathophysiological effects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR-mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identified a SNP within the EGFR promoter that modulates MR-induced EGFR expression. In RNA-sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to differential expression of cardiac ion channels, especially of the T-type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone- and EGF-responsiveness of CACNA1H expression was confirmed in HL-1 cells by Western blot and by measuring peak current density of T-type calcium channels. Aldosterone-induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T-type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL-1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an effect on HL-1 cell diameter, and the extent of this regulation seems to depend on the SNP-216 (G/T) genotype. This suggests that the EGFR may be an intermediate for MR-mediated cardiovascular changes and that SNP analysis can help identify subgroups of patients that will benefit most from MR antagonists.
Assuntos
Canais de Cálcio Tipo T/genética , Receptores ErbB/genética , Hipertrofia/genética , Receptores de Mineralocorticoides/genética , Aldosterona/genética , Animais , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/patologia , Linhagem Celular , Feminino , Genótipo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , RatosRESUMO
ATP, which is released under pathological conditions and is considered a damage-associated molecular pattern (DAMP), activates P2X7 receptors (P2X7Rs), trimeric plasma membrane ion channels selective for small cations. P2X7Rs are partners in NOD-like receptor containing a pyrin (NLRP3) inflammasome activation and promoters of tumor cell growth. P2X7R overstimulation triggers the ATP-dependent opening of a nonselective plasma membrane pore, known as a 'macropore', which allows fluxes of large hydrophilic molecules. The pathophysiological functions of P2X7R are thought to be dependent on activation of this conductance pathway, yet its molecular identity is unknown. Recent reports show that P2X7R permeability to organic solutes is an early and intrinsic property of the channel itself. A better understanding of P2X7R-dependent changes in plasma membrane permeability will allow a rationale development of novel anti-inflammatory and anticancer drugs.
Assuntos
Alarminas/genética , Permeabilidade da Membrana Celular/genética , Neoplasias/genética , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/metabolismo , Alarminas/metabolismo , Proliferação de Células/genética , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neoplasias/patologia , Transdução de SinaisRESUMO
P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1-P2X7) that form homo- and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X41-384 and C-terminally truncated fluorescent P2X71-408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration-response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.
RESUMO
One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca(2+) concentration ([Ca(2+)]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca(2+)]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca(2+)-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca(2+)]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K(+) concentration or by application of the Na(+) ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K(+) ionophore valinomycin, by extracellular Mg(2+), which is known to inhibit P2X7R-dependent effects, or by replacing Na(+) by the less P2X7R-permeable Tris(+) ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors.
Assuntos
Linfócitos B/imunologia , Núcleo Celular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Criança , Gramicidina/farmacologia , Humanos , Ativação Linfocitária , Magnésio/metabolismo , Fármacos Neuromusculares Despolarizantes/farmacologia , Potássio/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptor Cross-Talk , Valinomicina/farmacologiaRESUMO
Purinergic P2X(1) and P2X(7) receptors are co-expressed in several cell types such as lymphocytes or epithelial cells. Here we examined whether these two P2X subtypes interact with each other in a manner that results in a mutual alteration of their electrophysiologic behaviour. Furthermore, since specific pharmacological tools are needed to assign distinct effects to a particular receptor subtype in native cells, we assessed a series of compounds for their capacity to separate individual current components in cells that co-expressed both receptor subtypes. In Xenopus oocytes, co-expression neither changed the time courses of activation, desensitization and deactivation nor recovery from desensitization when compared to oocytes that express either hP2X(1) or hP2X(7) receptors alone. A selective activation of hP2X(7) receptors was achieved with benzoyl-benzoyl-ATP, which did not activate P2X(1) receptor currents. P2X(7) receptors could also be selectively activated by ATP when co-applied with 1 microM NF449, a suramin derivative, which is 100,000 fold more potent in blocking P2X(1) than P2X(7) receptors. alphabeta-methylene-ATP, a reportedly hP2X(1) receptor-specific agonist, as well as oxidized-ATP, brilliant blue or KN62, reported hP2X(7) receptor antagonists, were found to be ineffective in separating hP2X(1) receptor current from the P2X(7) current. The best way for a selective activation of the hP2X(1) receptor component in cells co-expressing the P2X(7) receptor is the application of low concentrations of ATP (< 1 microM) or the addition of Mg2+ when using higher concentrations of ATP.