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1.
Alcohol Alcohol ; 54(5): 497-502, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31535696

RESUMO

AIMS: The development of novel and more effective medications for alcohol use disorder (AUD) is an important unmet medical need. Drug repositioning or repurposing is an appealing strategy to bring new therapies to the clinic because it greatly reduces the overall costs of drug development and expedites the availability of treatments to those who need them. Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, is a drug used clinically to treat hyperuricemia and gout due to its activity as an inhibitor of the kidneys' organic anion transporter that reclaims uric acid from urine. Probenecid also inhibits pannexin1 channels that are involved in purinergic neurotransmission and inflammation, which have been implicated in alcohol's effects and motivation for alcohol. Therefore, we tested the effects of probenecid on alcohol intake in rodents. METHODS: We tested the effects of probenecid on operant oral alcohol self-administration in alcohol-dependent rats during acute withdrawal as well as in nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice. RESULTS: Probenecid reduced alcohol intake in both dependent and nondependent rats and in the DID paradigm in mice without affecting water or saccharin intake, indicating that probenecid's effect was selective for alcohol and not the result of a general reduction in reward. CONCLUSIONS: These results raise the possibility that pannexin1 is a novel therapeutic target for the treatment of AUD. The clinical use of probenecid has been found to be generally safe, suggesting that it can be a candidate for drug repositioning for the treatment of AUD.


Assuntos
Consumo de Bebidas Alcoólicas/tratamento farmacológico , Alcoolismo/tratamento farmacológico , Conexinas/antagonistas & inibidores , Sistemas de Liberação de Medicamentos/métodos , Etanol/administração & dosagem , Proteínas do Tecido Nervoso/antagonistas & inibidores , Probenecid/uso terapêutico , Adjuvantes Farmacêuticos/farmacologia , Adjuvantes Farmacêuticos/uso terapêutico , Consumo de Bebidas Alcoólicas/metabolismo , Consumo de Bebidas Alcoólicas/psicologia , Alcoolismo/metabolismo , Alcoolismo/psicologia , Animais , Conexinas/metabolismo , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Probenecid/farmacologia , Ratos , Ratos Wistar , Autoadministração
2.
Artigo em Inglês | MEDLINE | ID: mdl-31136852

RESUMO

Copper is a metal that participates in several essential reactions in living organisms, and it has been used as an inflammatory inducing agent in zebrafish larvae. In this study, we evaluated the effect P2X7 receptor and/or pannexin channel 1 (PANX-1) blockage in this inflammation model. To perform the experiments, 7 dpf larvae were exposed to 10 µM of copper and treated with 100 µM probenecid, PANX-1 inhibitor, and/or 300 nM A740003, a P2X7R selective antagonist. Larvae survival was assessed up to 24 h after treatments. The evaluation of larvae behavior was evaluated after acute (4 h) and chronic (24 h) exposure. The parameters of locomotor activity measured were: mobile time, average speed, distance and turn angle. We analyzed the gene expression of the P2X7 receptor, PANX1a and PANX1b channels and interleukins IL-10 and IL-1b after 24 h of treatment. Treatments did not decrease larval survival in the time interval studied. Changes in larvae locomotion were observed after the longest time of exposure to copper and the treatment with probenecid was able to reverse part of the effects caused by copper. No significant difference was observed in the oxidative stress assays and probenecid and copper treatment decrease partially PANX1a gene expression groups. The data presented herein shows the relevance of the blockage of P2X7-PANX-1 in copper-induced inflammation.


Assuntos
Conexinas/genética , Cobre/toxicidade , Inflamação/induzido quimicamente , Receptores Purinérgicos P2X7/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Acetamidas/farmacologia , Animais , Conexinas/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/mortalidade , Interleucina-10/genética , Interleucina-1beta/genética , Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Estresse Oxidativo , Probenecid/farmacologia , Antagonistas do Receptor Purinérgico P2X/toxicidade , Quinolinas/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores
3.
Int J Mol Sci ; 20(10)2019 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-31109150

RESUMO

Gap junction (GJ) channels in invertebrates have been used to understand cell-to-cell communication in vertebrates. GJs are a common form of intercellular communication channels which connect the cytoplasm of adjacent cells. Dysregulation and structural alteration of the gap junction-mediated communication have been proven to be associated with a myriad of symptoms and tissue-specific pathologies. Animal models relying on the invertebrate nervous system have exposed a relationship between GJs and the formation of electrical synapses during embryogenesis and adulthood. The modulation of GJs as a therapeutic and clinical tool may eventually provide an alternative for treating tissue formation-related diseases and cell propagation. This review concerns the similarities between Hirudo medicinalis innexins and human connexins from nucleotide and protein sequence level perspectives. It also sets forth evidence of computational techniques applied to the study of proteins, sequences, and molecular dynamics. Furthermore, we propose machine learning techniques as a method that could be used to study protein structure, gap junction inhibition, metabolism, and drug development.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Animais , Simulação por Computador , Conexinas/análise , Conexinas/antagonistas & inibidores , Junções Comunicantes/química , Humanos , Aprendizado de Máquina , Modelos Biológicos , Sistema Nervoso/química , Sistema Nervoso/metabolismo , Conformação Proteica
4.
Inflammation ; 42(3): 1082-1092, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30725252

RESUMO

Acute brain dysfunction and the following neurological manifestation are common complications in septic patients, which are associated with increased morbidity and mortality. However, the therapeutic strategy of this disorder remains a major challenge. Given the emerging role of a clinically approved drug, probenecid (PRB) has been recently identified as an inhibitor of pannexin 1 (PANX1) channel, which restrains extracellular ATP release-induced purinergic pathway activation and inflammatory response contributing to diverse pathological processes. In this study, we explored whether PRB administration attenuated neuroinflammatory response and cognitive impairment during sepsis. In mice suffered from cecal ligation and puncture (CLP)-induced sepsis, treatment with PRB improved memory retention and lessened behavioral deficits. This neuroprotective effect was coupled with restricted overproduction of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and interleukin (IL)-1ß in the hippocampus. Since this damped neuroinflammation was replicated by inhibition of ATP release, it suggested that PANX1 channel modulates a purinergic-related pathway contributing to the neurohistological damage. Therefore, we identified PRB could be a promising therapeutic approach for the therapy of cerebral dysfunction of sepsis.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Probenecid/farmacologia , Sepse/tratamento farmacológico , Adjuvantes Farmacêuticos , Animais , Córtex Cerebral/fisiopatologia , Transtornos Cognitivos/prevenção & controle , Conexinas/metabolismo , Inflamação/prevenção & controle , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Probenecid/uso terapêutico , Sepse/complicações
5.
Theriogenology ; 122: 144-149, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30268031

RESUMO

In the domestic cat, nuclear maturation and embryo development after vitrification of immature oocytes have been obtained but developmental competence after warming remains low. It has been reported that during folliculogenesis, the association and communication between the oocyte and the surrounding cumulus cells through connexin-based gap junctions is essential for normal oocyte and follicular development. Gap junctions result from the head-to-head interaction of two hemichannels; however, there is always a population of hemichannels not incorporated into gap junctions. These unopposed hemichannels are normally closed but may open under certain stress conditions, potentially also during vitrification and warming, turning them into toxic pores inducing cell injury and cell death. The aim of our study was to test whether inhibiting connexin 37 (Cx37) and connexin 43 (Cx43) channels with the connexin-targeting peptide Gap26 during vitrification and warming of cat immature cumulus-oocyte-complexes (COCs) could improve oocyte maturation and competence of resultant blastocysts derived by parthenogenetic activation. In the first experiment, our immunostainings confirmed the presence of Cx43 protein in the cytoplasm of immature cat oocytes and in the plasma membranes of cumulus cells. In the second experiment, COCs were randomly divided in three different groups: a control group (control), a group vitrified without Gap26 (vitrified) and a group vitrified with Gap26 (vitrified-peptide). The maturation rate was checked and oocytes from all three different experimental groups were parthenogenetically activated and cultured in vitro until day 8. After vitrification and warming, 49% of the oocytes in the control group matured, while this was 8% and 19% in the vitrified and vitrified-peptide groups, respectively. Compared to the vitrified group, oocytes in the vitrified-peptide group had significantly larger maturation rates. No blastocysts were detected at day 8 in the vitrified group, while 2% and 13% of the oocytes further developed to blastocyst at day 8 in the vitrified-peptide and control non-vitrified group, respectively. We conclude that the use of Gap26 in vitrification and warming media to vitrify immature cat oocytes improves maturation success and allows such oocytes to reach the blastocyst stage (2%) at day 8 after parthenogenetic activation.


Assuntos
Gatos , Conexinas/antagonistas & inibidores , Criopreservação/veterinária , Oócitos/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Blastocisto/citologia , Criopreservação/métodos , Desenvolvimento Embrionário , Junções Comunicantes/química , Junções Comunicantes/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/crescimento & desenvolvimento , Estresse Fisiológico , Vitrificação
6.
J Neurosci ; 38(35): 7713-7724, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30037834

RESUMO

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.


Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Líquido Cefalorraquidiano/fisiologia , Concentração de Íons de Hidrogênio , Hipotálamo/citologia , Mecanorreceptores/fisiologia , Neurônios/fisiologia , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Potenciais de Ação , Animais , Conexinas/antagonistas & inibidores , Conexinas/fisiologia , Feminino , Junções Comunicantes/fisiologia , Lampreias , Masculino , Movimento (Física) , Técnicas de Patch-Clamp , Estimulação Física , Somatostatina/análise , Estresse Mecânico , Terceiro Ventrículo , Ácido gama-Aminobutírico/análise
7.
PLoS One ; 13(7): e0200705, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30016355

RESUMO

Leonurine has been reported to play an important role in ameliorating cognitive dysfunction, inhibiting ischemic stroke, and attenuating perihematomal edema and neuroinflammation in intracerebral hemorrhage. However, the exact mechanism and potential molecular targets of this effect remain unclear. Thus, in this study we investigated the neuroprotective effects of leonurine on hypoxia ischemia injury and explored the underlying mechanisms. An in vitro model of oxygen-glucose deprivation (OGD)-induced PC12 cells was established to mimic ischemic-like conditions. Cell viability, apoptosis, Cx36 and pCaMKII/CaMKII expression levels were evaluated after treatment with leonurine. The Cx36-selective antagonist mefloquine and CaMKII Inhibitor KN-93 were used to investigate the neuroprotective effect of leonurine on and the involvement of Cx36/CaMKII in this process. The results revealed that cell viability decreased and cell apoptosis and the protein expression of Cx36 and pCaMKII/CaMKII increased in the OGD-induced PC12 cells. Leonurine significantly increased cell viability and decreased cell apoptosis and the protein expression of Cx36 and pCaMKII/CaMKII in the OGD-induced PC12 cells. The specific inhibitor of Cx36 and CaMKII displayed similar protective effects. Moreover, the inhibition of Cx36 reduced pCaMKII levels and the ratio of pCaMKII/CaMKII in the OGD-induced PC12 cells, and vice versa. Taken together, these results suggest that leonurine might have a protective effect on OGD-induced PC12 cells through targeting the Cx36/CaMKII pathway. Thus, leonurine appears to have potential as a preventive or therapeutic drug against ischemic-induced neuronal injury.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Conexinas/metabolismo , Ácido Gálico/análogos & derivados , Glucose , Fármacos Neuroprotetores/farmacologia , Oxigênio , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Conexinas/antagonistas & inibidores , Ácido Gálico/farmacologia , Mefloquina/farmacologia , Células PC12 , Ratos
9.
J Am Soc Nephrol ; 29(7): 1887-1899, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29866797

RESUMO

Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation.Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI.Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro, Panx1-deficient proximal tubule cells released less and retained more ATP under hypoxic stress.Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Conexinas/antagonistas & inibidores , Conexinas/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/prevenção & controle , Trifosfato de Adenosina/metabolismo , Animais , Antiulcerosos/farmacologia , Células da Medula Óssea/metabolismo , Carbenoxolona/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular , Células Epiteliais/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle
10.
Int J Mol Sci ; 19(6)2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29865195

RESUMO

Since their characterization more than five decades ago, gap junctions and their structural proteins-the connexins-have been associated with cancer cell growth. During that period, the accumulation of data and molecular knowledge about this association revealed an apparent contradictory relationship between them and cancer. It appeared that if gap junctions or connexins can down regulate cancer cell growth they can be also implied in the migration, invasion and metastatic dissemination of cancer cells. Interestingly, in all these situations, connexins seem to be involved through various mechanisms in which they can act either as gap-junctional intercellular communication mediators, modulators of signalling pathways through their interactome, or as hemichannels, which mediate autocrine/paracrine communication. This complex involvement of connexins in cancer progression is even more complicated by the fact that their hemichannel function may overlap with other gap junction-related proteins, the pannexins. Despite this complexity, the possible involvements of connexins and pannexins in cancer progression and the elucidation of the mechanisms they control may lead to use them as new targets to control cancer progression. In this review, the involvements of connexins and pannexins in these different topics (cancer cell growth, invasion/metastasis process, possible cancer therapeutic targets) are discussed.


Assuntos
Antineoplásicos/farmacologia , Carcinogênese , Conexinas/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Animais , Antineoplásicos/uso terapêutico , Comunicação Celular , Conexinas/antagonistas & inibidores , Conexinas/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transdução de Sinais
11.
Mol Brain ; 11(1): 28, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29793524

RESUMO

A high-fat diet (HFD) causes obesity by promoting excessive energy intake, and simultaneously, by disturbing the timing of energy intake. Restoring the feeding pattern is sufficient to prevent HFD-induced obesity in mice. However, the molecular mechanism(s) underlying HFD-induced feeding pattern disturbances remain elusive. Saturated fatty acids activate microglia and cause hypothalamic inflammation. Activated microglia cause neuroinflammation, which spreads via inflammatory cytokines and gap-junction hemichannels. However, the role of gap-junction hemichannels in HFD-induced obesity remains unaddressed. We used a novel, central-acting connexin inhibitor, INI-0602, which has high affinity for gap junction hemichannels and does not affect the induction of inflammatory cytokines. We analyzed ad libitum feeding behavior and locomotor activity in mice that were fed normal chow (NC), a HFD with elevated saturated fatty acids (SFAs), or a HFD with very high SFAs. We found that HFD feeding induced acute hyperphagia, mainly during the light cycle. Feeding pattern disturbances were more pronounced in mice that consumed the HFD with very high SFAs than in mice that consumed the HFD with elevated SFAs. When INI-0602 was administered before the HFD was introduced, it blocked the feeding pattern disturbance, but not locomotor activity disturbances; moreover, it prevented subsequent diet-induced obesity. However, when INI-0602 was administered after the HFD had disturbed the feeding pattern, it failed to restore the normal feeding pattern. Therefore, we propose that SFAs in HFDs played a major role in disrupting feeding patterns in mice. Moreover, the feeding pattern disturbance required the function of central, gap junction hemichannels at the initiation of a HFD. However, altering hemichannel function after the feeding pattern disturbance was established had no effect. Thus, preventing the occurrence of a feeding pattern disturbance by blocking the hemichannel pathway was associated with the prevention of the HFD-induced obesity in mice.


Assuntos
Conexinas/antagonistas & inibidores , Comportamento Alimentar , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Obesidade/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Conexinas/metabolismo , Citocinas/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Atividade Motora/efeitos dos fármacos , Obesidade/patologia
12.
Am J Rhinol Allergy ; 32(4): 217-227, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29676177

RESUMO

Background Airway mucociliary transport is an important function for the clearance of inhaled foreign particulates in the respiratory tract. The present study aimed at investigating the regulatory mechanism of acetylcholine (Ach)-induced ciliary beat of the human nasal mucosa in ex vivo. Methods The inferior turbinate mucosa was collected from patients with chronic hypertrophic rhinitis during endoscopic surgery. The mucosa was cut into thin strips, and ciliary movement was observed under a phase-contrast light microscope with a high-speed digital video camera. The sample was alternatively subjected to scanning electron microscopic observation. Results Cilia on the turbinate epithelium were well preserved at the ultrastructural level. The baseline ciliary beat frequency (CBF) was 6.45 ± 0.32 Hz. CBF was significantly increased by stimulation with 100 µM Ach and 100 µM adenosine triphosphate. The Ach-induced CBF increase was completely inhibited by removing extracellular Ca2+. Significant inhibition of the Ach-induced CBF was also observed by the addition of 1 µM atropine, 40 µM 2-aminoethoxydiphenyl borate (inositol trisphosphate [IP3] receptor antagonist), 10 µM carbenoxolone (pannexin-1 blocker), 1 mM probenecid (pannexin-1 blocker), 100 µM pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid (P2X antagonist), and 300 µM flufenamic acid (connexin blocker). Meanwhile, 30 nM bafilomycin A1 (vesicular transport inhibitor) did not inhibit the Ach-induced CBF increase. CONCLUSIONS: These results indicate that the regulatory mechanism of the Ach-induced ciliary beat is dependent on extracellular Ca2+ and involves the muscarinic Ach receptor, IP3 receptor, pannexin-1 channel, purinergic P2X receptor, and connexin channel. We proposed a tentative intracellular signaling pathway of the Ach-induced ciliary beat, in which the pannexin-1-P2X unit may play a central role in ciliary beat regulation.


Assuntos
Cílios/fisiologia , Conexinas/metabolismo , Depuração Mucociliar/fisiologia , Mucosa Nasal/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X/metabolismo , Rinite/imunologia , Acetilcolina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carbenoxolona/farmacologia , Células Cultivadas , Doença Crônica , Conexinas/antagonistas & inibidores , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Transdução de Sinais , Conchas Nasais/patologia , Adulto Jovem
13.
Neuro Oncol ; 20(7): 885-896, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29106645

RESUMO

A rational treatment strategy for glioma, the most common primary central nervous system tumor, should focus on early invasive growth and resistance to current therapeutics. Connexin 43 (Cx43), a gap junction protein, plays important roles not only in the development of the central nervous system and but also in the progression of glioma. The different structural domains of Cx43, including extracellular loops, transmembrane domains, and an intracellular carboxyl terminal, have distinct functions in the invasion and proliferation of gliomas. Targeting these domains of Cx43, which is expressed in distinct patterns in the heterogeneous glioma cell population, can inhibit tumor cell invasion and new tumor formation. Thus, this review summarizes the structural characteristics of Cx43, the effects of regulating different Cx43 domains on the biological characteristics of glioma cells, intervention strategies targeting different domains of Cx43, and future research directions.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Conexinas/antagonistas & inibidores , Glioma/tratamento farmacológico , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Terapia de Alvo Molecular , Prognóstico , Domínios Proteicos
14.
Hum Mol Genet ; 27(1): 80-94, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29077882

RESUMO

Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Doença de Charcot-Marie-Tooth/metabolismo , Conexinas/genética , Conexinas/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Canais de Cálcio/genética , Doença de Charcot-Marie-Tooth/genética , Conexinas/antagonistas & inibidores , Conexinas/química , Citosol/metabolismo , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Modelos Moleculares , Técnicas de Patch-Clamp , Células de Schwann/metabolismo , Transfecção
15.
Hypertension ; 71(1): 151-159, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29084879

RESUMO

Coordination of vascular smooth muscle cell tone in resistance arteries plays an essential role in the regulation of peripheral resistance and overall blood pressure. Recent observations in animals have provided evidence for a coupling between adrenoceptors and Panx1 (pannexin-1) channels in the regulation of sympathetic nervous control of peripheral vascular resistance and blood pressure; however, evidence for a functional coupling in humans is lacking. We determined Panx1 expression and effects of treatment with the pharmacological Panx1 channel inhibitor probenecid on the vasoconstrictor response to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle. Probenecid treatment increased (P<0.05) leg vascular conductance at baseline by ≈15% and attenuated (P<0.05) the leg vasoconstrictor response to arterial infusion of tyramine (α1- and α2-adrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition of α1-adrenoceptors prevented the probenecid-induced increase in baseline leg vascular conductance, but did not alter the effect of probenecid on the vascular response to tyramine. No differences with probenecid treatment were detected in the forearm. These observations provide the first line of evidence in humans for a functional role of Panx1 channels in setting resting tone via α1-adrenoceptors and in the constrictive effect of noradrenaline via α2-adrenoceptors, thereby contributing to the regulation of peripheral vascular resistance and blood pressure in humans.


Assuntos
Artérias , Pressão Sanguínea , Conexinas , Extremidades/irrigação sanguínea , Músculo Liso Vascular , Proteínas do Tecido Nervoso , Receptores Adrenérgicos , Sistema Nervoso Simpático , Vasoconstrição , Inibidores da Captação Adrenérgica/farmacologia , Adulto , Artérias/efeitos dos fármacos , Artérias/patologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Conexinas/antagonistas & inibidores , Conexinas/metabolismo , Humanos , Masculino , Músculo Liso Vascular/inervação , Músculo Liso Vascular/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Probenecid/farmacologia , Receptores Adrenérgicos/classificação , Receptores Adrenérgicos/metabolismo , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Tiramina/farmacologia , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasoconstritores/farmacologia
16.
Circ Res ; 122(4): 606-615, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29237722

RESUMO

RATIONALE: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. OBJECTIVE: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. METHODS AND RESULTS: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. CONCLUSIONS: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.


Assuntos
Anti-Hipertensivos/farmacologia , Conexinas/antagonistas & inibidores , Diuréticos/farmacologia , Hipertensão/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Espironolactona/farmacologia , Animais , Anti-Hipertensivos/uso terapêutico , Arteríolas/efeitos dos fármacos , Conexinas/metabolismo , Diuréticos/uso terapêutico , Células HEK293 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Espironolactona/uso terapêutico
17.
Mol Cell Endocrinol ; 472: 97-106, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29208420

RESUMO

The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a KATP channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca2+ channel activity, cytosolic Ca2+ concentration ([Ca2+]i) and insulin secretion. Mefloquine was tested in the concentration range of 5-50 µM 25 µM mefloquine was as effective as 500 µM tolbutamide to depolarize the plasma membrane of beta cells, but did not induce action potentials. Rather, it abolished tolbutamide-induced action potentials and the associated increase of [Ca2+]i. In the range of 5-50 µM mefloquine inhibited voltage-dependent Ca2+ currents in primary beta cells as effectively as 1 µM nisoldipine, a specific blocker of L-type Ca2+ channels. The Ca2+ channel opening effect of Bay K8644 was completely antagonized by mefloquine. Likewise, the increase of [Ca2+]i and of insulin secretion stimulated by 40 mM KCl, but not that by 30 mM glucose was antagonized by 50 µM mefloquine. Neither at 5 µM nor at 50 µM did mefloquin stimulate insulin secretion at basal glucose. In conclusion, mefloquine blocks KATP channels and L-type Ca2+ channels in pancreatic beta cells in the range from 5 to 50 µM. Thus it inhibits depolarization-induced insulin secretion, but in the presence of a stimulatory glucose concentration additional effects of mefloquine, possibly on intracellular Ca2+ mobilization, and the metabolic amplification by glucose permit a sustained rate of secretion.


Assuntos
Canais de Cálcio/metabolismo , Conexinas/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Mefloquina/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Conexinas/metabolismo , Di-Hidropiridinas/farmacologia , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Nisoldipino/farmacologia , Cloreto de Potássio/farmacologia , Tolbutamida/farmacologia
18.
PLoS One ; 12(11): e0188135, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29145453

RESUMO

BACKGROUND: Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis. METHODS: Thioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5-10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5-10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA. RESULTS: Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1. CONCLUSIONS: These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Antivirais/farmacologia , Conexinas/antagonistas & inibidores , Modelos Animais de Doenças , Cirrose Hepática/prevenção & controle , Fígado/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Dermatopatias/prevenção & controle , Pele/efeitos dos fármacos , Tenofovir/farmacologia , Animais , Conexinas/fisiologia , Relação Dose-Resposta a Droga , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Pele/metabolismo
19.
Curr Pharm Des ; 23(33): 4958-4968, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28982320

RESUMO

BACKGROUND: In Alzheimer's disease (AD), modification of astrocytic properties is a well-known and documented fact, but their involvement in pathophysiology has only been examined in recent years. One distinct hallmark of AD is reactive gliosis which are represented in microglial and astrocytic phenotype changes. This reactive gliosis has been associated with changes in the expression and function of connexins. Connexins are proteins that can form gap junction channels and hemichannels, and in a disease context, have shown increased expression in astrocytes that contact amyloid plaques in vivo. Amyloid plaques are aggregates of the amyloid-beta protein, which present in the AD brain in patients and in animal models. METHODS: Murine AD models demonstrate changes in connexin channel activity which mirror in cell culture systems treated with amyloid-beta peptide. This has been closely studied in the familial AD mouse model APPSwe/ PS1dE9 where the implications of connexin channel functions have been examined. RESULTS: These studies demonstrate that while gap junctional communication was unaffected, hemichannel activation could be detected in the astrocytes of hippocampal slices containing amyloid-beta plaques. Most critically, the activation of hemichannels is associated with the release of gliotransmitters (such as ATP and glutamate) which results in the maintenance of a high intracellular free Ca2+ concentration within astrocytes, which initiates the start of a vicious cycle. Strategies that target astroglial connexin hemichannels include the knocking out of the connexin 43 gene in astrocytes of the APPSwe/PS1dE9 mice, as well as using various pharmacological tools. This results in the decrease of gliotransmitter release and the alleviation of neuronal damage. This includes the reduction of oxidative stress and neuritic dystrophies in neurons that are typically associated with plaque formation in the hippocampus. Concusion: In this review, we summarize recent findings on astroglial connexin channels in the neurodegenerative process of Alzheimer's disease, and discuss how this can be a strategy in AD treatment to block the activity of hemichannels in astrocytes.


Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Conexinas/antagonistas & inibidores , Conexinas/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Doença de Alzheimer/tratamento farmacológico , Animais , Astrócitos/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Fármacos Neuroprotetores/administração & dosagem
20.
Pharmacol Rev ; 69(4): 396-478, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28931622

RESUMO

Connexins are ubiquitous channel forming proteins that assemble as plasma membrane hemichannels and as intercellular gap junction channels that directly connect cells. In the heart, gap junction channels electrically connect myocytes and specialized conductive tissues to coordinate the atrial and ventricular contraction/relaxation cycles and pump function. In blood vessels, these channels facilitate long-distance endothelial cell communication, synchronize smooth muscle cell contraction, and support endothelial-smooth muscle cell communication. In the central nervous system they form cellular syncytia and coordinate neural function. Gap junction channels are normally open and hemichannels are normally closed, but pathologic conditions may restrict gap junction communication and promote hemichannel opening, thereby disturbing a delicate cellular communication balance. Until recently, most connexin-targeting agents exhibited little specificity and several off-target effects. Recent work with peptide-based approaches has demonstrated improved specificity and opened avenues for a more rational approach toward independently modulating the function of gap junctions and hemichannels. We here review the role of connexins and their channels in cardiovascular and neurovascular health and disease, focusing on crucial regulatory aspects and identification of potential targets to modify their function. We conclude that peptide-based investigations have raised several new opportunities for interfering with connexins and their channels that may soon allow preservation of gap junction communication, inhibition of hemichannel opening, and mitigation of inflammatory signaling.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Conexinas/antagonistas & inibidores , Conexinas/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Animais , Doenças Cardiovasculares/tratamento farmacológico , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Humanos , Doenças do Sistema Nervoso/tratamento farmacológico , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos
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