RESUMO
In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(ß2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.
Assuntos
Células Quimiorreceptoras/metabolismo , Receptores Nicotínicos/metabolismo , Respiração , Traqueia/fisiologia , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Paladar , Traqueia/citologia , Traqueia/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, ß2, and ß4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 µM) induced a dose-dependent increase of the transepithelial short-circuit current (EC(50): 14.6 µM). The nicotine-induced effect (I(NIC)) was attenuated by mecamylamine (25 µM, apical) and methyllycaconitine (1 µM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 µM) revealed apical and basolateral location of the receptors. I(NIC) was not affected by the sodium channel inhibitor amiloride (10 µM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172 (20 µM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM, apical), the Na(+)/K(+)/2Cl(-) cotransporter inhibitor bumetanide (200 µM, basolateral), the potassium channel inhibitor Ba(2+) (5 mM, basolateral), and 4.4'-diisothiocyanatostilbene-2.2'-disulfonate (100 µM, apical), indicating a contribution of Ca(2+)-activated chloride channels and potassium channels. Removal of extracellular Na(+) (apical) or Ca(2+) (apical) did not influence I(NIC) but reduced the DMPP effect. Experiments with the Ca(2+)-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP(3)) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 µM, apical) decreased I(NIC), indicating a nicotine-mediated increase of intracellular Ca(2+) and cAMP levels involving the IP(3) signaling pathway. These findings indicate the activity of Ca(2+)-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl(-) and K(+) transport in tracheal epithelium.
Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Epitélio/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Receptores Nicotínicos/metabolismo , Traqueia/metabolismo , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Epitélio/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/farmacologia , Receptores Nicotínicos/genética , Transdução de Sinais/efeitos dos fármacos , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Traqueia/efeitos dos fármacosRESUMO
Dyspnoea is frequently observed in cancer cachectic patients. Little is known whether this is accompanied by structural or functional alterations of the lung. We hypothesized that in analogy to calorie restriction cancer cachexia leads to loss of alveolar surface area and surfactant. Mice were subjected to subcutaneous injection of Lewis lung carcinoma cells (tumour group, TG) or saline (control group, CG). Twenty-one days later blood samples and the lungs were taken. Using design-based stereology, the alveolar surface area and the lamellar body (Lb) content were quantified. Messenger RNA expression of surfactant proteins, ABCA3 and various growth factors was investigated by quantitative RT-PCR. Intraalveolar surfactant subtype composition was analyzed by differential centrifugation. TG mice showed reduced body weight and anaemia but no reduction of lung volume or alveolar surface area. The volume of Lb was significantly reduced and mRNA levels of ABCA3 transporter tended to be lower in TG versus CG. Surfactant protein expression and the ratio between active and inactive intraalveolar surfactant subtypes were not altered in TG. Growth factor mRNA levels were not different between CG and TG lungs but the tumour expressed growth factor mRNA. Vascular endothelial growth factor was significantly enhanced in blood plasma. The present study demonstrates structural alterations of the lung associated with cancer cachexia. These include reduction of Lb content despite normal intraalveolar surfactant and alveolar surface area. The pulmonary phenotype of the cancer cachectic mouse differs from the calorie restricted mouse possibly due to growth factors released from the tumour tissue.
Assuntos
Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Carcinoma Pulmonar de Lewis/patologia , Feminino , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Fator A de Crescimento do Endotélio Vascular/sangue , Redução de Peso/fisiologiaRESUMO
Epithelial cells (ECs) are usually hyporesponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram-negative bacteria, is impeded, at least in part, by intracellular sequestration of its receptor, Toll-like receptor-4 (TLR4). In this study, using human bronchial ECs (hBECs) as a model of mucosal epithelium, we tested the hypothesis that the human LPS-binding, membrane-active cationic host defense peptide cathelicidin LL-37 augments epithelial response to LPS by facilitating its delivery to TLR4-containing intracellular compartments. We found that LL-37 significantly increases uptake of LPS by ECs with subsequent targeting to cholera toxin subunit B-labeled structures and lysosomes. This uptake is peptide specific, dose and time dependent, and involves the endocytotic machinery, functional lipid rafts, and epidermal growth factor receptor signaling. Cathelicidin-dependent LPS internalization resulted in significant increased release of the inflammatory cytokines IL-6 and IL-8. This indicates that, in ECs, this peptide may replace LPS-binding protein functions. In polarized ECs, the effect of LL-37 was restricted to the basolateral compartment of the epithelial membrane, suggesting that LL-37-mediated activation of ECs by LPS may be relevant to disease conditions associated with damage to the epithelial barrier. In summary, our study identified a novel role of LL-37 in host-microbe interactions as a host factor that licenses mucosal ECs to respond to LPS.
Assuntos
Catelicidinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Peptídeos Catiônicos Antimicrobianos , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Microscopia Confocal , Transdução de Sinais/efeitos dos fármacosRESUMO
Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Complexo do Signalossomo COP9 , Proteínas de Ciclo Celular/genética , Humanos , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Células NIH 3T3 , Peptídeo Hidrolases/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitinação , Proteína com ValosinaRESUMO
Cholinergic bronchoconstriction is mediated by M(2) and M(3) muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-ß-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R(-/-) and M3R(-/-) mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.
Assuntos
Brônquios/fisiologia , Broncoconstrição/fisiologia , Cavéolas/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Sequência de Bases , Brônquios/ultraestrutura , Broncoconstrição/genética , Cavéolas/ultraestrutura , Caveolinas/genética , Caveolinas/metabolismo , Primers do DNA/genética , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Miócitos de Músculo Liso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M2/deficiência , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Transdução de SinaisRESUMO
Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.
Assuntos
Células Dendríticas/patologia , Hipersensibilidade/patologia , Inflamação/patologia , Sistema Respiratório/patologia , Linfócitos T/patologia , Animais , Antígeno CD11c/genética , Divisão Celular , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Modelos Animais de Doenças , Hipersensibilidade/imunologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Fibras Nervosas/patologia , Neurônios/imunologia , Neurônios/patologia , Ovalbumina , Sistema Respiratório/imunologia , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
BACKGROUND: An increased susceptibility of micro-vascular endothelial cells to apoptosis is considered to be an initial event leading to atherosclerosis. Parathyroid hormone-related peptide (PTHrP) is known to protect endothelial cells against apoptosis by the regulation of the anti-apoptotic gene bcl-2. As tissue inhibitor of metalloproteinase (TIMP-1) expression is regulated by bcl-2, we hypothesized that endothelial expression of PTHrP also regulates the expression of TIMP-1. METHODS: The steady state mRNA expressions of bcl-2, bax, TIMP-1, and TIMP-2 were analyzed by real-time RT-PCR and their protein expression by immunoblotting. The tissue distribution of PTHrP was investigated in cryosections of hearts from normotensive and hypertensive rats. RESULTS: Phenylephrine, an alpha(1)-adrenoceptor agonist, increased the expression of PTHrP, bcl-2, and TIMP-1. Transfection of endothelial cells with oligonucleotides directed against PTHrP attenuated this effect. Antisense transfection and TGF-beta(1) (10 ng/ml) decreased the expression of PTHrP, bcl-2, TIMP-1, and TIMP-2, but not that of bax. Endothelial cells were identified as the main source of PTHrP in the heart. Endothelial cells in hearts from spontaneously hypertensive rats showed reduced staining with a PTHrP antibody compared to control normotensive hearts. CONCLUSIONS: These data suggests that the down-regulation of PTHrP favours atherosclerosis in chronic pressure overload.
Assuntos
Vasos Coronários/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Apoptose , Células Cultivadas , Regulação para Baixo , Masculino , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fenilefrina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Antissenso/metabolismo , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: After transplantation, passenger leukocytes move to lymphoid organs of the recipient. These cells appear to initiate allograft rejection, but they also might be involved in tolerance induction. MATERIALS AND METHODS: Orthotopic left lung transplantation was performed in the Dark Agouti to Lewis rat strain combination with no immunosuppression. Recipient spleens were removed at intervals of 24 h until day 6 after transplantation. For comparison, spleens from renal allograft recipients were analysed. Donor-derived major histocompatibility complex (MHC) class II antigens were detected by monoclonal antibody OX76. In double-staining experiments with antibodies specific for leukocyte subpopulations, their localisation and identity was analysed. RESULTS: OX76-positive leukocytes were already detected in recipient spleens on day 1 post-transplantation. They increased in number until day 3 and decreased in number thereafter. Most of them were localised in splenic follicles and expressed the B cell variant of CD45R and IgG. Cell surface antigens typical for other leukocyte subpopulations were not detected. In the spleens of renal allograft recipients, only few donor-derived cells were seen. CONCLUSION: After lung transplantation, numerous MHC class II-positive B cells migrate to the splenic follicles of the recipient. These cells might, in part, be responsible for immunologic differences observed between renal and pulmonary allografts.
Assuntos
Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Transplante de Pulmão/imunologia , Baço/imunologia , Doadores de Tecidos , Animais , Linfócitos B/patologia , Ensaios de Migração de Leucócitos , Rejeição de Enxerto/patologia , Antígenos de Histocompatibilidade Classe II/análise , Transplante de Rim/imunologia , Transplante de Rim/patologia , Antígenos Comuns de Leucócito , Transplante de Pulmão/patologia , Contagem de Linfócitos , Masculino , Prognóstico , Ratos , Ratos Endogâmicos , Baço/patologia , Transplante HomólogoRESUMO
In ciliated airway epithelial cells endothelial nitric oxide synthase as well as several other membrane bound proteins are located in the apical cell pole. To date, mechanisms that serve to target and to keep these proteins in this region are unknown. Endothelial nitric oxide synthase is known to target to caveolae by interaction with caveolin-1 or caveolin-3. Since caveolin-1 is found only in a subpopulation of ciliated cells at the basolateral cell membrane, we examined if caveolin-3 could be responsible for the apical localization of endothelial nitric oxide synthase in ciliated cells. We used real-time RT-PCR, laser-assisted microdissection, Western blotting and double-labeling immunohistochemistry to examine the presence of caveolin-3 in the airway epithelium of the rat. Indeed, we found caveolin-3-mRNA as well as protein in ciliated cells throughout the trachea and the bronchial tree. Caveolin-3-immunoreactivity was confined to the apical region and was colocalized with endothelial nitric oxide synthase and the high affinity choline transporter in a compartment distinct from the plasma membrane at the light microscopic level. No caveolae were found in the apical plasma membrane of ciliated cells but a tubulovesicular network was present in the apical region that reached up to the basal bodies of the cilia and was in close contact with mitochondria. Co-immunoprecipitation of caveolin-3 with endothelial nitric oxide synthase verified that both proteins interact in airway ciliated cells. These findings indicate that caveolin-3 is responsible to keep endothelial nitric oxide synthase in a membrane compartment in the apical region of ciliated cells.
Assuntos
Caveolina 3/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Traqueia/metabolismo , Animais , Anticorpos Monoclonais , Sequência de Bases , Western Blotting , Caveolina 3/genética , Caveolina 3/imunologia , Cílios/metabolismo , Primers do DNA/genética , Células Epiteliais/metabolismo , Imuno-Histoquímica , Microscopia Eletrônica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/citologiaRESUMO
Fast excitatory synaptic transmission in sympathetic ganglia is mediated by nicotinic acetylcholine receptors (nAChRs). Although it is known that the nAChR alpha7-subunit occurs in sympathetic ganglia, the expression of the recently cloned subunit alpha10 (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002) has not been analyzed. Until now, functional receptors containing alpha10-subunits have been found only in combination with alpha9-subunits (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002). The alpha9-subunit exhibits a restricted expression pattern, whereas the alpha10-subunit is expressed more widely. This broad distribution resembles more closely that known for subunit alpha7 than for subunit alpha9. On this background, we investigated the distribution of nAChR subunits alpha7, alpha9, and alpha10 in rat sympathetic ganglia and studied a possible interaction between subunit alpha7 and potential partners by double-labeling immunofluorescence and fluorescence resonance energy transfer (FRET) (Kam et al., 1995; Jares-Erijman and Jovin, 2003).
Assuntos
Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Animais , Hibridização In Situ , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND: Caveolae are involved in diverse cellular functions such as signal transduction, cholesterol homeostasis, endo- and transcytosis, and also may serve as entry sites for microorganisms. Hence, their occurrence in epithelium of the airways might be expected but, nonetheless, has not yet been examined. METHODS: Western blotting, real-time quantitative PCR analysis of abraded tracheal epithelium and laser-assisted microdissection combined with subsequent mRNA analysis were used to examine the expression of cav-1 and cav-2, two major caveolar coat proteins, in rat tracheal epithelium. Fluorescence immunohistochemistry was performed to locate caveolae and cav-1 and -2 in the airway epithelium of rats, mice and humans. Electron-microscopic analysis was used for the identification of caveolae. CLSM-FRET analysis determined the interaction of cav-1alpha and cav-2 in situ. RESULTS: Western blotting and laser-assisted microdissection identified protein and transcripts, respectively, of cav-1 and cav-2 in airway epithelium. Real-time quantitative RT-PCR analysis of abraded tracheal epithelium revealed a higher expression of cav-2 than of cav-1. Immunoreactivities for cav-1 and for cav-2 were co-localized in the cell membrane of the basal cells and basolaterally in the ciliated epithelial cells of large airways of rat and human. However, no labeling for cav-1 or cav-2 was observed in the epithelial cells of small bronchi. Using conventional double-labeling indirect immunofluorescence combined with CLSM-FRET analysis, we detected an association of cav-1alpha and -2 in epithelial cells. The presence of caveolae was confirmed by electron microscopy. In contrast to human and rat, cav-1-immunoreactivity and caveolae were confined to basal cells in mice. Epithelial caveolae were absent in cav-1-deficient mice, implicating a requirement of this caveolar protein in epithelial caveolae formation. CONCLUSION: These results show that caveolae and caveolins are integral membrane components in basal and ciliated epithelial cells, indicating a crucial role in these cell types. In addition to their physiological role, they may be involved in airway infection.
Assuntos
Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Mucosa Respiratória/metabolismo , Animais , Brônquios/citologia , Brônquios/metabolismo , Brônquios/ultraestrutura , Cavéolas/fisiologia , Caveolina 1/genética , Caveolina 2/genética , Feminino , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Mucosa Respiratória/ultraestrutura , Traqueia/citologia , Traqueia/metabolismo , Traqueia/ultraestruturaRESUMO
Caveolin-1 (Cav-1) is highly expressed in alveolar epithelial type I (AE1) and endothelial cells of the alveolar region of the lung. Interestingly, alveolar epithelial type II (AE2) cells that are progenitors of the AE1 cells do not express Cav-1. We investigated whether genetic Cav-1 deficiency alters the phenotype of AE2 cells and their microenvironment using stereology. Total number, mean volume, and subcellular composition of the AE2 cells were not altered in Cav-1(-/-) when compared with wild-type mice. The alveolar septa were thickened and contained a significantly greater volume of extracellular matrix. Thus, AE2 cells as progenitors of AE1 cells are not critically involved in the severe pulmonary phenotype in Cav-1-deficient mice.
Assuntos
Células Epiteliais Alveolares/citologia , Caveolina 1/deficiência , Microambiente Celular/fisiologia , Células Epiteliais Alveolares/metabolismo , Animais , Cavéolas/metabolismo , Caveolina 1/genética , Matriz Extracelular/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
During acute rejection, numerous pro-inflammatory and cytotoxic monocytes accumulate in the vasculature of experimental renal allografts. Arrestins (ARRBs) are cellular regulators of inflammation, but nothing is known about their expression during rejection. Intravascular mononuclear graft leukocytes were isolated 4 days after kidney transplantation. ARRB1 and ARRB2 mRNA expression was reduced in blood leukocytes from allografts undergoing acute rejection, whereas on the protein level only ARRB2 was changed. Flow cytometry and confocal microscopy revealed ARRB1 and ARRB2 expression by monocytes and T cells, with a selective decrease in ARRB2 expression in monocytes during acute rejection. I-κB directly interacted with ARRB2 and the levels of both proteins strongly correlated. Concomitantly, the mRNA expression of NF-κB targeted genes increased. Our results suggest that activation of blood monocytes in renal isografts is dampened by high ARRB2 levels. During acute rejection, ARRB2 levels are reduced and classical monocyte activation is enabled via NF-κB activation.
Assuntos
Arrestinas/metabolismo , Rejeição de Enxerto/imunologia , Transplante de Rim , Monócitos/metabolismo , Transplante Homólogo/patologia , Doença Aguda , Animais , Arrestinas/genética , Arrestinas/imunologia , Separação Celular , Regulação para Baixo/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Humanos , Masculino , Modelos Animais , Monócitos/imunologia , Monócitos/patologia , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
T-cell intracellular antigen (TIA)-1 and TIA-1-related protein (TIAR) are mRNA-binding proteins that can aggregate within granules under specific stress conditions. In this study, we analyzed TIAR/TIA-1 aggregation under different hypoxic conditions, and studied the effects on the hypoxia-inducible factor (HIF)-1α in different cancer cell lines. Under acute and pronounced hypoxic conditions TIAR/TIA-1 co-aggregated to granules and positive co-staining with eIF3η marker suggested these to represent stress granules. In parallel, HIF-1α expression was blocked in cells displaying TIAR/TIA-1 granules. Silencing of TIAR and TIA-1 caused upregulation of HIF-1α expression, as demonstrated by western blot, immunocytochemistry and HIF-1-dependent reporter gene expression. Additionally, a critical region of the 3' end of the untranslated HIF-1α mRNA with possible adenosine-uridine-rich elements (AREs) was coupled to the luciferase reporter gene, causing downregulation of expression. Employing this reporter construct, inhibition of TIAR by siRNA attenuated the inhibitory cis-effect of this ARE-sequence. Furthermore, immunohistochemical analysis of A549 cell tumor xenografts revealed a nearly complementary expression of HIF-1α and TIAR reflecting the control of HIF-1α expression by TIAR as revealed in the cell culture studies. In sum, rapid and severe hypoxia caused co-aggregation of TIAR/TIA-1 and these proteins suppressed HIF-1α expression.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Proteínas de Ligação a Poli(A)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Antígeno-1 Intracelular de Células T , Transplante HeterólogoRESUMO
Quantitative information about the innervation is essential to analyze the structure-function relationships of organs. So far, there has been no unbiased stereological tool for this purpose. This study presents a new unbiased and efficient method to quantify the total length of axons in a given reference volume, illustrated on the left ventricle of the mouse heart. The method is based on the following steps: 1) estimation of the reference volume; 2) randomization of location and orientation using appropriate sampling techniques; 3) counting of nerve fiber profiles hit by a defined test area within an unbiased counting frame on paraffin sections stained immunohistochemically for protein gene product 9.5; 4) electron microscopic estimation of the mean number of axon profiles contained in one nerve fiber profile; 5) estimation of the degree of tissue shrinkage of specimens in paraffin; and 6) calculation of the total axon length within the reference volume, taking tissue shrinkage into account. In a set of five mouse hearts, the total length of axons ramifying between cardiomyocytes ranged between approximately 50 and 100 m, with a mean of 75.98 m (SD 23.73). The time required for the microscopical analysis was approximately 8 h/animal for an experienced observer. Using antibodies specific for different neuron subtypes and immunoelectron microscopy, this method is also suited to estimate the total axon length of neurons expressing different transmitters. This new and efficient method is particularly useful when structural remodeling takes place and is suspected to involve gain or loss of axons.
Assuntos
Axônios , Técnicas Citológicas , Ventrículos do Coração/inervação , Neurônios , Animais , Axônios/química , Axônios/ultraestrutura , Feminino , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Neurônios/química , Neurônios/ultraestrutura , Inclusão em Parafina , Reprodutibilidade dos Testes , Ubiquitina Tiolesterase/análiseRESUMO
BACKGROUND: Mucociliary clearance in the airways is driven by the coordinated beating of ciliated cells. Classical neuromediators such as noradrenalin and acetylcholine increase ciliary beat frequency and thus cilia-driven transport. Despite the fact that the neuromediator serotonin is ciliostimulatory in invertebrates and has been implied in releasing acetylcholine from the airway epithelium, its role in regulating cilia function in vertebrate airways is not established. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of serotonin on ciliary beat frequency and cilia-driven particle transport in the acutely excised submerged mouse trachea and determined the sources of serotonin in this tissue by immunohistochemistry. Serotonin (100 microM) increased cilary beat frequency (8.9+/-1.2 Hz to 17.0+/-2.7 Hz) and particle transport speed (38.9+/-4.6 microm/s to 83.4+/-8.3 microm/s) to an extent that was comparable to a supramaximal dose of ATP. The increase in particle transport speed was totally prevented by methysergide (100 microM). Blockade of muscarinic receptors by atropine (1 microM) did not reduce the effect of serotonin, although it was effective in preventing the increase in particle transport speed mediated by muscarine (100 microM). Immunohistochemistry demonstrated serotonin in mast cells pointing towards mast cells and platelets as possible endogenous sources of serotonin. CONCLUSIONS/SIGNIFICANCE: These results indicate that serotonin is a likely endogenous mediator that can increase cilia-driven transport independent from acetylcholine during activation of mast cells and platelets.
Assuntos
Acetilcolina/fisiologia , Cílios/fisiologia , Serotonina/fisiologia , Traqueia/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Atropina/farmacologia , Cílios/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traqueia/efeitos dos fármacosRESUMO
Calcitonin gene-related peptide (CGRP)-alpha is expressed in heart ventricles in sensory nerves and cardiomyocytes. It modifies inotropism and induces ischaemic preconditioning. This study investigates the effect of CGRP-alpha on the contractile responsiveness of isolated adult ventricular rat cardiomyocytes and the effect of chronic hypertension on this interaction. Cardiomyocytes were isolated and paced at 0.5-2.0 Hz. Cell shortening was recorded via a line camera with a reading frame of 500 Hz. CGRP-alpha exerted a dual effect on cardiomyocytes with a positive contractile effect at 10nM and a negative contractile effect at 10 pM. CGRP-alpha(8-37), a calcitonin receptor-like receptor (CRLR) antagonist, attenuated the positive contractile effect. H89, a protein kinase A antagonist, converted the positive contractile effect into a negative contractile effect. The negative contractile effect was converted again back to a positive contractile effect in the presence of l-nitro arginine. In cardiomyocytes isolated from spontaneously hypertensive rats (SHR) the mRNA expression of CRLR and the receptor-associated modifier protein (RAMP)-2 were lower. However, on the protein level CLRL was up-regulated, RAMP2 expression remained unchanged, and eNOS expression was down-regulated in these cells. These cells responded with a pure positive contractile response. In Langendorff preparations, CGRP-alpha slightly reduced the rate pressure product in hearts from normotensive rats but it caused an increase in hearts from SHR. In conclusion, it is shown that CGRP-alpha exerts dual effects on cardiomyocytes favouring the negative contractile effect at very low concentrations. This effect is compensated in chronic pressure-overloaded hearts and converted into a positive inotropism.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Função Ventricular/efeitos dos fármacos , Animais , Pressão Sanguínea/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoquinolinas/farmacologia , Proteínas de Membrana/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Proteína 2 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/antagonistas & inibidores , Sulfonamidas/farmacologia , Função Ventricular/fisiologiaRESUMO
Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.
Assuntos
Cavéolas/metabolismo , Endocitose/fisiologia , Produtos do Gene tat/metabolismo , Pulmão/citologia , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Animais , Células Cultivadas , DNA/metabolismo , Produtos do Gene tat/química , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Pulmão/fisiologia , Oligopeptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , TransgenesRESUMO
It is pivotal to identify protein-protein interaction in situ to understand protein function. Conventional methods to determine the interaction of proteins destruct tissue or are applicable to cell culture only. To identify association of proteins in cells in tissue, we adapted indirect double-labeling immunofluorescence and combined it with conventional confocal laser scanning microscopy (CLSM) to measure fluorescence resonance energy transfer (FRET). As a model system, we chose caveolin-1alpha and caveolin-2, two major components of endothelial caveolae, and examined their interaction in the endothelium of vessels in fixed tissues of laboratory animals and human glomus tumors. Several methodological aspects were examined. Measuring the absolute increase in fluorescence (DeltaIF) was superior compared to determining the relative FRET efficiency, because it is more robust against small increases of fluorescence during measurements that results from unavoidable minimal crossreactivity of the secondary antibodies. Both, sequential and simultaneous incubation of secondary antibodies result in robust and reliable increases in DeltaIF. If incubated sequentially, however, the acceptor-labeled secondary antibody should be applied first. The size of the secondary reagent (F(ab')2 vs whole antibody) has no major influence. In conclusion, CLSM-FRET can measure close spatial association of proteins in situ and can be applied to human surgical material.