RESUMO
A proton (H+) channel, Otopetrin 1 (OTOP1) is an acid sensor in the sour taste receptor cells. Although OTOP1 is known to be activated by extracellular acid, no posttranslational modification of OTOP1 has been reported. As one of the posttranslational modifications, glycosylation is known to modulate many ion channels. In this study, we investigated whether OTOP1 is glycosylated and how the glycosylation affects OTOP1 function. Pharmacological and enzymatic examinations (using an N-glycosylation inhibitor, tunicamycin and peptide: N-glycanase F [PNGase F]) revealed that overexpressed mouse OTOP1 was N-glycosylated. As the N-glycans were Endoglycosidase H (Endo H)-sensitive, they were most likely high-mannose type. A site-directed mutagenesis approach revealed that both two asparagine residues (N238 and N251) in the third extracellular loop between the fifth transmembrane region and the sixth transmembrane region (L5-6) were the glycosylation sites. Prevention of the glycosylations by the mutations of the asparagine residues or by tunicamycin treatment diminished the whole-cell OTOP1 current densities. The results of cell surface biotinylation assay showed that the prevention of the glycosylations reduced the surface expression of OTOP1 at the plasma membrane. These results indicate that mouse OTOP1 is N-glycosylated at N238 and N251, and that the glycosylations are necessary for OTOP1 to show the maximum degree of H+ current densities at the plasma membrane through promoting its targeting to the plasma membrane. These findings on glycosylations of OTOP1 will be a part of a comprehensive understanding on the regulations of OTOP1 function.
RESUMO
Persistent primitive hypoglossal artery is a relatively rare anatomical variation and a type of persistent carotid-basilar anastomosis. Acute internal carotid artery occlusion associated with persistent primitive hypoglossal artery is rare, and atherothrombotic occlusion is extremely rare. We present a case of acute atherothrombotic internal carotid artery occlusion associated with persistent primitive hypoglossal artery that was successfully treated by endovascular treatment. A 70-year-old male with a history of left internal carotid artery stenosis was transferred to our hospital by ambulance because of abnormal behaviors and aphasia. He was diagnosed with cerebral infarction and left internal carotid artery occlusion. Left carotid angiography revealed the persistent primitive hypoglossal artery arising from the cervical internal carotid artery and complete internal carotid artery occlusion distal to the origin of the persistent primitive hypoglossal artery. Therefore, we performed endovascular treatment. Mechanical thrombectomy was performed under minimal flow arrest with consideration of brain ischemia causing coma. After additional balloon angioplasty, recanalization was achieved, and the patient's symptoms improved. During the 1.5-year follow-up period, no recurrence or restenosis was observed. This report provides evidence that atherosclerotic internal carotid artery stenosis associated with persistent primitive hypoglossal artery can occur even distal to the origin of the persistent primitive hypoglossal artery and that the lesion may become acutely occluded, leading to acute stroke. Endovascular treatment considering brain ischemia was effective in this case.
Assuntos
Isquemia Encefálica , Doenças das Artérias Carótidas , Estenose das Carótidas , Masculino , Humanos , Idoso , Artéria Carótida Interna/diagnóstico por imagem , Artéria Carótida Interna/cirurgia , Estenose das Carótidas/complicações , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/cirurgia , Doenças das Artérias Carótidas/complicações , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/cirurgia , Isquemia Encefálica/complicaçõesRESUMO
Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells.
Assuntos
Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas , Animais , Humanos , Camundongos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células HEK293 , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/metabolismo , Estereocílios/metabolismoRESUMO
Otopetrin 1 (OTOP1) is a proton (H+) channel which detects acidic stimuli in sour taste receptor cells and plays some sort of role in the formation of otoconia in the inner ear. Although it is known that zinc ion (Zn2+) inhibits OTOP1, Zn2+ requires high concentrations (mM order) to inhibit OTOP1 sufficiently, and no other inhibitors have been found. Therefore, to identify a novel inhibitor, we screened a chemical library (LOPAC1280) by whole-cell patch clamp recordings, measuring proton currents of heterologously-expressed mouse OTOP1. From the screening, we found that reactive blue 2 inhibited OTOP1 currents. Further evaluations of three analogues of reactive blue 2 revealed that cibacron blue 3G-A potently inhibited OTOP1 currents. Cibacron blue 3G-A inhibited OTOP1 currents in a concentration-dependent manner, and its 50% inhibitory concentration (IC50) and the Hill coefficient were 5.0 µM and 1.1, respectively. The inhibition of OTOP1 currents by cibacron blue 3G-A was less affected by extracellular anion compositions, membrane potentials, and low pH than the inhibition by Zn2+. These results suggest that the inhibition of OTOP1 by cibacron blue 3G-A is neither likely to be a pore-blocking inhibition nor a competitive inhibition. Furthermore, our findings revealed that cibacron blue 3G-A can be used as a novel inhibitor of OTOP1 especially under the conditions in which OTOP1 activity is evaluated such as low pH.
Assuntos
Prótons , Triazinas , Camundongos , Animais , Triazinas/farmacologia , Proteínas de MembranaRESUMO
Posterior reversible encephalopathy syndrome, an acute onset neurological syndrome, is among the conditions that must be differentiated from stroke. Herein, we report a rare case of posterior reversible encephalopathy syndrome mimicking subacute ischemic stroke. A 68-year-old man was transferred by ambulance to our hospital because of visual disturbance. He showed left homonymous hemianopsia. Magnetic resonance imaging (diffusion-weighted imaging and fluid-attenuated inversion recovery imaging) revealed high signal intensity in the right occipital lobe. We suspected subacute cerebral infarction. After admission, he developed cortical blindness and increased blood pressure. Fluid-attenuated inversion recovery imaging revealed high signal intensity and elevated apparent diffusion coefficient values in the bilateral occipital lobes. We diagnosed the patient with posterior reversible encephalopathy syndrome. Antihypertensive treatment improved his clinical symptoms. Careful imaging assessment, including of changes over time, is important for diagnosing posterior reversible encephalopathy syndrome.
RESUMO
Astrocytes are glial cells with numerous fine processes which are important for the functions of the central nervous system. The activation of ß-adrenoceptors induces process formation of astrocytes via cyclic AMP (cAMP) signaling. However, the role of α-adrenoceptors in the astrocyte morphology has not been elucidated. Here, we examined it by using cultured astrocytes from neonatal rat spinal cords and cortices. Exposure of these cells to noradrenaline and the ß-adrenoceptor agonist isoproterenol increased intracellular cAMP levels and induced the formation of processes. Noradrenaline-induced process formation was enhanced with the α1-adrenoceptor antagonist prazosin and α2-adrenoceptor antagonist atipamezole. Atipamezole also enhanced noradrenaline-induced cAMP elevation. Isoproterenol-induced process formation was not inhibited by the α1-adrenoceptor agonist phenylephrine but was inhibited by the α2-adrenoceptor agonist dexmedetomidine. Dexmedetomidine also inhibited process formation induced by the adenylate cyclase activator forskolin and the membrane-permeable cAMP analog dibutyryl-cAMP. Moreover, dexmedetomidine inhibited cAMP-independent process formation induced by adenosine or the Rho-associated kinase inhibitor Y27632. In the presence of propranolol, noradrenaline inhibited Y27632-induced process formation, which was abolished by prazosin or atipamezole. These results demonstrate that α-adrenoceptors inhibit both cAMP-dependent and -independent astrocytic process formation.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Receptores Adrenérgicos alfa/fisiologia , Receptores Adrenérgicos beta/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Dexmedetomidina/farmacologia , Imidazóis/farmacologia , Isoproterenol/farmacologia , Norepinefrina/farmacologia , Prazosina/farmacologia , Ratos Wistar , Transdução de SinaisRESUMO
Direct hybridization between the π-orbital of a conjugated molecule and metal electrodes is recognized as a new anchoring strategy to enhance the electrical conductance of single-molecule junctions. The anchor is expected to maintain direct hybridization between the conjugated molecule and the metal electrodes, and control the orientation of the molecule against the metal electrodes. However, fulfilling both requirements is difficult because multipodal anchors aiming at a robust contact with the electrodes often break the π-conjugation, thereby resulting in an inefficient carrier transport. Herein, a new tripodal anchor framework-a 7,7-diphenyl-7H-benzo[6,7]indeno[1,2-b]thiophene (PBIT) derivative-is developed. In this framework, π-conjugation is maintained in the molecular junction, and the tripodal structure makes the molecule stand upright on the metal electrode. Molecular conductance is measured by the break junction technique. A vector-based classification and first-principles transport calculations determine the single-molecule conductance of the tripodal-anchoring structure. The conductance of the PBIT-based molecule is higher than that of the tripodal anchor having sp3 carbon atoms in the carrier transport pathway. These results demonstrate that extending the π-conjugation to the tripodal leg is an effective strategy for enhancing the conductivities of single-molecule junctions.
RESUMO
Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca2+ concentration ([Ca2+]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca2+]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca2+ signaling, which is known to be involved in synaptic function. Na2S (150 µM), an H2S donor, produced a nontoxic increase in [Ca2+]i. The [Ca2+]i increase by Na2S was inhibited by Ca2+ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca2+, indicating that H2S releases Ca2+ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca2+ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca2+ signaling by decreasing Ca2+ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.
Assuntos
Trifosfato de Adenosina/farmacologia , Astrócitos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Medula Espinal/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Ratos Wistar , Medula Espinal/metabolismoRESUMO
Mechanical stimuli caused by sound waves are detected by hair cells in the cochlea through the opening of mechanoelectrical transduction (MET) channels. Transmembrane channel-like protein 1 (TMC1) has been revealed to be the pore-forming component of the MET channel. The two splice variants for mouse Tmc1 (mTmc1ex1 and mTmc1ex2) were reported to be expressed in the cochlea of infant mice, though only the sequence of mTmc1ex2 had been deposited in GenBank. However, due to the presence of an upstream open reading frame (uORF) and the absence of a typical Kozak sequence in mTmc1ex2, we questioned whether mTMC1 was translated from mTmc1ex2. Therefore, in this study, we evaluated which splice variant was protein-coding mRNA. Firstly, the results of RT-PCR and cDNA cloning of mTmc1 using mRNA isolated from the cochlea of five-week-old mice suggested that more Tmc1ex1 were expressed than mTmc1ex2. Secondly, mTMC1 was translated from mTmc1ex1 but not from mTmc1ex2 in a heterologous expression system. Finally, analyses using site-directed mutagenesis revealed that the uORF and the weak Kozak sequence in mTmc1ex2 prevented the translation of mTMC1 from mTmc1ex2. These results suggest that mTmc1ex1 plays a main role in the expression of mTMC1 in the mouse cochlea, and therefore, mTmc1ex1 should be the mRNA for mTMC1 hereafter.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Processamento Alternativo/genética , Animais , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Masculino , Mecanorreceptores/metabolismo , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Isoformas de Proteínas/genéticaRESUMO
Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.
Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ratos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genéticaRESUMO
Purinergic signaling via ATP and adenosine produced by astrocytes is one pathway underlying neuron-glia interactions in the central nervous system (CNS). In production of purines, extracellular metabolism of released purines via ecto-enzymes is important. The expression and activities of these enzymes are altered under pathological conditions. Production of fibroblast growth factor 2 (FGF2) is increased under pathological conditions, and this has various effects on astrocytes. Here, we investigated the effects of FGF2 on purine metabolism in cultured rat spinal cord astrocytes. Astrocytes rapidly metabolized purines added to the extracellular solution. FGF2 increased extracellular metabolism of AMP to adenosine and of adenosine to inosine by upregulating ecto-5'-nucleotidase and adenosine deaminase (ADA), respectively. ADA activity and protein were detected both in the cytosol and external solution of astrocytes, and their levels were markedly increased by FGF2. FGF2 also increased metabolism of endogenously released ATP, resulting in a transient increase in adenosine and substantial accumulation of extracellular inosine. Moreover, FGF2 increased ATP release by upregulating the activity of gap junction hemichannels. These data show that FGF2 regulates purine production in astrocytes and suggest that extracellular ADA released by astrocytes plays an important role in extracellular purine metabolism in the CNS.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Astrócitos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Purinas/metabolismo , Animais , Astrócitos/metabolismo , Células Cultivadas , Feminino , Masculino , Ratos Wistar , Medula Espinal/citologia , Regulação para CimaRESUMO
Xylazine, the classical α2 -adrenoceptor (α2 -AR) agonist, is still used as an analgesic and sedative in veterinary medicine, despite its low potency and affinity for α2 -ARs. Previous pharmacological studies suggested that the α2A -AR subtype plays a role in mediating the clinical effects of xylazine; however, these studies were hampered by the poor subtype-selectivity of the antagonists used and a lack of knowledge of their bioavailability in vivo. Here, we attempted to elucidate the role of the α2A -AR subtype in mediating the clinical effects of xylazine by comparing the analgesic and sedative effects of this drug in wild-type mice with those in α2A -AR functional knockout mice using the hot-plate and open field tests, respectively. Hippocampal noradrenaline turnover in both mice was also measured to evaluate the contribution of α2A -AR subtype to the inhibitory effect of xylazine on presynaptic noradrenaline release. In wild-type mice, xylazine (10 or 30 mg/kg) increased the hot-plate latency. Furthermore, xylazine (3 or 10 mg/kg) inhibited the open field locomotor activity and decreased hippocampal noradrenaline turnover. By contrast, all of these effects were abolished in α2A -AR functional knockout mice. These results indicate that the α2A -AR subtype is mainly responsible for the clinical effects of xylazine.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Analgésicos/farmacologia , Hipnóticos e Sedativos/farmacologia , Receptores Adrenérgicos alfa 2/metabolismo , Xilazina/farmacologia , Animais , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Norepinefrina/metabolismoRESUMO
H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+]i increase, and 3) rapid [Ca2+]i increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1ß augmented H2S-induced [Ca2+]i increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1ß treatment did not affect [Ca2+]i increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1ß treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+]i increase in IL-1ß-treated cells. These results suggest that IL-1ß augments H2S-induced [Ca2+]i increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Interleucina-1beta/farmacologia , Óxido Nítrico/metabolismo , Sulfetos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Citocinas/farmacologia , Interações Medicamentosas , Óxido Nítrico Sintase/antagonistas & inibidores , Cloreto de Potássio/farmacologia , Ratos , Sulfetos/farmacologia , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/antagonistas & inibidoresRESUMO
Mas-related G-protein coupled receptor B4 (MrgprB4) has been reported to be expressed in the dorsal root ganglion (DRG) neurons which detect stroking of hairy skin of mice. However, the mechanisms by which the MrgprB4 positive (+) neurons respond to adequate stimulus remain unsolved as it was also reported that electrophysiological analysis of cultured MrgprB4+ neurons did not reveal responses to mechanical stimuli. Contrary to the observation, however, in this study we show that the MrgprB4+ neurons functionally express a mechanically activated channel using DRG neurons dissociated from genetically-modified mice whose MrgprB4+ neurons express a red fluorescent protein. Hypotonicity-induced cell swelling increased intracellular Ca2+ concentrations ([Ca2+]i) of MrgprB4+ neurons. The [Ca2+]i increases were prevented by extracellular Ca2+ removal and by applications of nonselective Piezo channel blockers. Patch clamp analysis revealed that the MrgprB4+ neurons exhibited rapidly-adapting mechanically-activated currents. The MrgprB4+ neurons were stained with anti-Piezo2 antibody. These results raise the possibility that the MrgprB4+ neurons directly detect the stroking-like stimuli of hairy skin.
Assuntos
Gânglios Espinais/fisiologia , Canais Iônicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/fisiologia , Tato , Animais , Cálcio/metabolismo , Feminino , Gânglios Espinais/metabolismo , Masculino , Camundongos , Estimulação Física , Células Receptoras Sensoriais/metabolismoRESUMO
Epithelioid cells in the chicken thoracic aorta are chemoreceptor cells that release 5-HT in response to hypoxia. It is likely that these cells play a role in chemoreception similar to that of glomus cells in the carotid bodies of mammals. Recently, H2S was reported to be a key mediator of carotid glomus cell responses to hypoxia. The aim of the present study was to reveal the mechanism of action of H2S on 5-HT outflow from chemoreceptor cells in the chicken thoracic aorta. The 5-HT outflow induced by NaHS, an H2S donor, and Na2S3, a polysulfide, was measured by using a HPLC equipped with an electrochemical detector. NaHS (0.3-3mM) caused a concentration-dependent increase in 5-HT outflow, which was significantly inhibited by the removal of extracellular Ca(2+). 5-HT outflow induced by NaHS (0.3mM) was also significantly inhibited by voltage-dependent L- and N-type Ca(2+) channel blockers and a selective TRPA1 channel blocker. Cinnamaldehyde, a TRPA1 agonist, mimicked the secretory response to H2S. 5-HT outflow induced by Na2S3 (10µM) was also inhibited by the TRPA1 channel blocker. Furthermore, the expression of TRPA1 was localized to 5-HT-containing chemoreceptor cells in the aortic wall. These findings suggest that the activation of TRPA1 and voltage-dependent Ca(2+) channels is involved in H2S-evoked 5-HT release from chemoreceptor cells in the chicken aorta.
Assuntos
Aorta Torácica/efeitos dos fármacos , Células Quimiorreceptoras/efeitos dos fármacos , Células Epitelioides/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Serotonina/metabolismo , Sulfetos/farmacologia , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Animais , Aorta Torácica/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/efeitos dos fármacos , Canais de Cálcio Tipo N/metabolismo , Células Quimiorreceptoras/metabolismo , Galinhas , Relação Dose-Resposta a Droga , Células Epitelioides/metabolismo , Técnicas In Vitro , Masculino , Transdução de Sinais/efeitos dos fármacos , Sulfetos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Chemoreceptor cells aggregating in clusters in the chicken thoracic aorta contain 5-hydroxytryptamine (5-HT) and have voltage-dependent ion channels and nicotinic acetylcholine receptors, which are characteristics typically associated with neurons. The aim of the present study was to investigate the effects of 5-HT uptake inhibitors, fluvoxamine, fluoxetine and clomipramine (CLM), and amphetamine derivatives, p-chloroamphetamine (PCA) and methamphetamine (MET), on endogenous 5-HT outflow from the isolated chick thoracic aorta in vitro. 5-HT was measured by using a HPLC system with electrochemical detection. The amphetamine derivatives and 5-HT uptake inhibitors caused concentration-dependent increases in endogenous 5-HT outflow. PCA was about ten times more effective in eliciting 5-HT outflow than MET. The 5-HT uptake inhibitors examined had similar potency for 5-HT outflow. PCA and CLM increased 5-HT outflow in a temperature-dependent manner. The outflow of 5-HT induced by PCA or 5-HT uptake inhibitors was independent of extracellular Ca(2+) concentration. The 5-HT outflow induced by CLM, but not that by PCA, was dependent on the extracellular NaCl concentration. These results suggest that the 5-HT uptake system of 5-HT-containing chemoreceptor cells in the chicken thoracic aorta has characteristics similar to those of 5-HT-containing neurons in the mammalian central nervous system (CNS).
Assuntos
Aorta Torácica/efeitos dos fármacos , Metanfetamina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , p-Cloroanfetamina/farmacologia , Animais , Aorta Torácica/metabolismo , Galinhas , Clomipramina/farmacologia , Fluoxetina/farmacologia , Fluvoxamina/farmacologia , Técnicas In Vitro , MasculinoRESUMO
Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K(+) (KATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both KATP and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both KATP and TRPA1 channels. RIN14B cells showed several intracellular Ca(2+) concentration ([Ca(2+)]i) responses to NaHS (300 µM), an H2S donor, i.e., inhibition of spontaneous Ca(2+) oscillations (37%), inhibition followed by [Ca(2+)]i increase (24%), and a rapid increase in [Ca(2+)]i (25%). KATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca(2+)]i increases, which were inhibited by extracellular Ca(2+) removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening KATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Canais KATP/efeitos dos fármacos , Células Secretoras de Somatostatina/efeitos dos fármacos , Sulfetos/farmacologia , Canais de Cátion TRPC/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Sulfeto de Hidrogênio/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Canais KATP/genética , Canais KATP/metabolismo , Cinética , Potenciais da Membrana , Ratos , Serotonina/metabolismo , Células Secretoras de Somatostatina/metabolismo , Sulfetos/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismoRESUMO
Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors.
Assuntos
Adenosina Quinase/antagonistas & inibidores , Analgésicos/farmacologia , Morfolinas/farmacologia , Neurônios Motores/efeitos dos fármacos , Dor Nociceptiva/tratamento farmacológico , Pirimidinas/farmacologia , Medula Espinal/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina/metabolismo , Antagonistas do Receptor A1 de Adenosina/farmacologia , Adenosina Desaminase/metabolismo , Adenosina Quinase/metabolismo , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Motores/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiopatologia , Dor Nociceptiva/fisiopatologia , Reflexo/efeitos dos fármacos , Reflexo/fisiologia , Medula Espinal/fisiopatologia , Técnicas de Cultura de Tecidos , Xantinas/farmacologiaRESUMO
Alpha-2A adrenergic receptor (AR) subtype plays an important role in the analgesic effect of α2-AR agonists. Here, we examined the effects of α2-AR agonists, dexmedetomidine and xylazine, on spinal synaptic transmission in newborn C57BL/6J and α2A-AR mutant mice. Spinal reflex potentials, the monosynaptic reflex potential (MSR) and the slow ventral root potential (sVRP), were measured in isolated spinal cords. The compound action potential was measured in isolated lumbar nerve. Dexmedetomidine and xylazine suppressed both the MSR and sVRP in a concentration-dependent manner. In α2A-AR mutant mice, sVRP suppression by dexmedetomidine was greatly weakened, while that by xylazine (30-100µM) showed only slight attenuation. A high concentration (300µM) of xylazine completely suppressed the sVRP, even in α2A-AR mutant mice spinal cords, and also suppressed the compound action potential. MSR suppression by these α2-AR agonists had no difference between wild-type and α2A-AR mutant mice. These results suggest that sVRP suppression by dexmedetomidine and xylazine is mainly mediated by α2A-AR. In addition, a high concentration of xylazine inhibits conduction of the action potential, which is not mediated by α2A-AR. α2-AR is not responsible for the dexmedetomidine- and xylazine-mediated inhibition of the MSR.
Assuntos
Neurônios Adrenérgicos/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Dexmedetomidina/farmacologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Xilazina/farmacologia , Potenciais de Ação , Neurônios Adrenérgicos/metabolismo , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Ratos Wistar , Receptores Adrenérgicos alfa 2/deficiência , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Reflexo/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/metabolismo , Fatores de TempoRESUMO
Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.