RESUMO
HIV is a major public health issue, and infection of CD4(+) T lymphocytes is one of its key features. Whereas several cellular proteins have been identified that facilitate viral infection and replication, the role of hemichannels in these processes has not been fully characterized. We now show that the HIV isolates, R5 and X4, induced a transient-early (5-30 min) and a later, persistent (48-120 h) opening of Panx1 hemichannels, which was dependent on the binding of HIV to CD4 and CCR5/CXCR4 receptors. Blocking Panx1 hemichannels by reducing their opening or protein expression inhibited HIV replication in CD4(+) T lymphocytes. Thus, our findings demonstrate that Panx1 hemichannels play an essential role in HIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Conexinas/fisiologia , HIV/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Conexina 43/fisiologia , Humanos , Receptores CCR5/fisiologia , Receptores CXCR4/fisiologia , Replicação ViralRESUMO
Neurological deficits in the offspring caused by human maternal hypothyroxinemia are thought to be irreversible. To understand the mechanism responsible for these neurological alterations, we induced maternal hypothyroxinemia in pregnant rats. Behavior and synapse function were evaluated in the offspring of thyroid hormone-deficient rats. Our data indicate that, when compared with controls, hypothyroxinemic mothers bear litters that, in adulthood, show prolonged latencies during the learning process in the water maze test. Impaired learning capacity caused by hypothyroxinemia was consistent with cellular and molecular alterations, including: 1) lack of increase of phosphorylated c-fos on the second day of the water maze test; 2) impaired induction of long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area 1 of the hippocampus Ammon's horn stratum radiatum, despite normal responses for input/output experiments; 3) increase of postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartic acid receptor subunit 1, and tyrosine receptor kinase B levels in brain extracts; and 4) significant increase of PSD-95 at the PSDs and failure of this molecule to colocalize with N-methyl-D-aspartic acid receptor subunit 1, as it was shown by control rats. Our findings suggest that maternal hypothyroxinemia is a harmful condition for the offspring that can affect key molecular components for synaptic function and spatial learning.
Assuntos
Transtornos Cognitivos/fisiopatologia , Hipotireoidismo/fisiopatologia , Aprendizagem em Labirinto/fisiologia , Complicações na Gravidez/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Tiroxina/deficiência , Fatores Etários , Animais , Transtornos Cognitivos/etiologia , Proteína 4 Homóloga a Disks-Large , Feminino , Hipotireoidismo/complicações , Imidazóis , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Fosforilação , Gravidez , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Percepção Espacial/fisiologia , Sinapses/fisiologia , Tiroxina/sangueRESUMO
Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Comunicação Celular/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Tamoxifeno/farmacologia , Tretinoína/farmacologia , Apoptose , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Conexina 26 , Conexinas , Humanos , Imuno-Histoquímica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Telomerase/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The Infectious Systemic Inflammatory Response syndrome and multiple organic dysfunction have common physiopathological mechanisms. Multiple organic dysfunction can be assessed using severity scores. AIM: To relate cytokine kinetics with a multiple organic dysfunction score during sepsis. MATERIAL AND METHODS: Tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL6) kinetics were studied in 25 patients with severe sepsis with less than 48 h of evolution and interleukin 1 beta (IL beta) kinetics was studied in 13 patients. Measurements were made at 0, 12, 24 and 48 hours after admission to the study, using an ELISA technique. These parameters were correlated with the Marshall multiple organic dysfunction score and survival. RESULTS: Mean age of study subjects was 70 years, the APACHE II score was 16.9 +/- 6 and the Marshall score was 6.8 +/- 3.6. Sepsis was of pulmonary origin in 56% of patients and intra abdominal in 32%. Mortality was 36%. TNF alpha increased during the study period (24.1 pg/ml initially and 37.8 pg/ml at 24 hours, with a slight posterior reduction, p < 0.02). These levels had no association with mortality or organic dysfunction. IL6 remained elevated during the first hours and had a tendency to decrease thereafter. Decreased patients had higher values than survivors (306 pg/ml and 55.4 pg/ml respectively, p = 0.011). Its values were tightly correlated with Marshall score, with the number of failing organs, with the presence of shock and with probability of dying during hospitalization. IL1 beta remained low and was not associated with clinical parameters. CONCLUSIONS: There is a tight correlation between the elevation of IL6 and the severity of the Systemic Inflammatory Response and mortality in these patients with sepsis.
Assuntos
Citocinas/metabolismo , Insuficiência de Múltiplos Órgãos/mortalidade , Sepse/mortalidade , APACHE , Idoso , Citocinas/sangue , Feminino , Mortalidade Hospitalar , Humanos , Interleucina-1/sangue , Interleucina-1/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Masculino , Insuficiência de Múltiplos Órgãos/metabolismo , Estudos Prospectivos , Sepse/metabolismo , Índice de Gravidade de Doença , Choque Séptico/metabolismo , Choque Séptico/mortalidade , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/mortalidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/efeitos dos fármacos , Interferon gama/farmacologia , Microglia/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Lesões Encefálicas/metabolismo , Comunicação Celular/efeitos dos fármacos , Conexina 43/deficiência , Junções Comunicantes/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microglia/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.
Assuntos
Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Timo/fisiologia , Animais , Conexinas/fisiologia , Células Epiteliais , Matriz Extracelular , Humanos , Imunidade Celular , Camundongos , RNA Mensageiro , Timo/citologiaRESUMO
Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system
Assuntos
Humanos , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Sistema Imunitário/citologia , Sistema Imunitário/fisiologia , Células da Medula Óssea/citologia , Comunicação Celular/fisiologia , Imunidade Celular/fisiologia , Células Estromais/fisiologiaRESUMO
We aimed at characterizing the receptor subtype and the signaling pathway involved in the inhibitory effect of neuropeptide Y on the release of endogenous noradrenaline from rat hypothalamus. Slices of hypothalamus were stimulated with two trains of electrical pulses, and the release of noradrenaline and nitric oxide was measured. The electrical stimulation of hypothalamic slices induced a consistent release of both endogenous noradrenaline and NO. Neuropeptide Y inhibited concentration dependently the stimulated noradrenaline release. Similarly, agonists for neuropeptide Y Y1, Y2 and Y5 receptors inhibited noradrenaline release, albeit with a potency lower than neuropeptide Y. GW1229, a selective neuropeptide Y Y1 receptor antagonist counteracted the effect of neuropeptide Y, but not that of PYY-(3-36), an agonist active at neuropeptide Y Y5 and Y2 receptors. These results indicate that the inhibitory effect of neuropeptide Y is likely mediated by several receptor subtypes, including neuropeptide Y Y1, Y5 and possibly Y2 receptors. One microM NPY significantly enhanced NO release induced by the electrical stimulation. NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, abolished NO release and blocked the inhibitory effect of neuropeptide Y on noradrenaline release. We conclude that nitric oxide participates in the signaling pathway of neuropeptide Y in the rat hypothalamus.
Assuntos
Hipotálamo/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Óxido Nítrico/metabolismo , Norepinefrina/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Animais , Estimulação Elétrica , Hipotálamo/metabolismo , Masculino , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismoRESUMO
Because hepatocytes communicate via gap junctions, it has been proposed that Ca2+ waves propagate through this pathway and in the process activate Ca2+-dependent cellular responses. We testedthis hypothesis by measuring vasopressin-induced glycogenolysis in short-term cultures of rat hepatocytes. A 15-min vasopressin (10(-8) M) stimulation induced a reduction of glycogen content that reached a maximum 1-3 h later. Gap junction blockers, octanol or 18alpha-glycyrrhetinic acid, reduced the effect by 70%. The glycogenolytic response induced by Ca2+ ionophore 8-bromo-A-21387, which acts on each hepatocyte, was not affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a synthetic peptide homologous to a domain of the extracellular loop 1 of the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced. Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP as a paracrine mediator. We propose that gap junctions allow for a coordinated vasopressin-induced glycogenolytic response despite the heterogeneity among hepatocytes.