RESUMO
BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Células Epiteliais/efeitos dos fármacos , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Hiperpotassemia/induzido quimicamente , Depuração Mucociliar/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Piperidinas/farmacologia , Administração por Inalação , Amilorida/efeitos adversos , Animais , Bloqueadores do Canal de Sódio Epitelial/efeitos adversos , Guanidinas , Cobaias , Técnicas In Vitro , Éteres Fenílicos/efeitos adversos , Piperidinas/efeitos adversos , Pirazinas , Ratos , Mucosa Respiratória/citologia , OvinosRESUMO
Increased numbers of macrophages and neutrophils in the lung is a key feature of chronic obstructive pulmonary disease (COPD). The major neutrophil chemotactic agent in the airways of COPD patients is leukotriene (LT)B(4) and is released by macrophages. The present study examines the role and mechanism of Ca(2+) in platelet-activating factor (PAF)-stimulated LTB(4) release from human lung macrophages. Macrophages were isolated from lung tissue of subjects undergoing lung resection surgery and monocyte-derived macrophages (MDM) were obtained from nonsmokers, smokers without obstruction and COPD patients. Cells were stimulated with PAF and LTB(4) release and [Ca(2+)](i) was measured. Lung macrophages and MDM released LTB(4) following stimulation with PAF (mean effective concentration: 0.08+/-0.06 microM (n = 5) versus 0.17+/-0.12 microM (n = 17), respectively). Compared with MDM, lung macrophages released approximately eight-fold more LTB(4). Neither smoking nor COPD altered MDM responses. PAF-stimulated LTB(4) release was abrogated by ethylene glycol tetraacetic acid suggesting a role for extracellular Ca(2+). This was substantiated by using store-operated channel blockers econazole, SK&F96365 and Gd(3+). However, econazole and SK&F96365 were more effective in MDM than lung macrophages. Neither LOE908 nor nifedipine could attenuate this response. These data suggest that platelet-activating factor-stimulated leukotriene B(4) release from human lung macrophages is mediated, in part, by Ca(2+) influx through receptor- but not voltage-operated Ca(2+) channels.
Assuntos
Canais de Cálcio/metabolismo , Leucotrieno B4/metabolismo , Macrófagos Alveolares/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Adulto , Área Sob a Curva , Bioensaio/métodos , Sinalização do Cálcio , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/metabolismoRESUMO
ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.
Assuntos
Trifosfato de Adenosina/farmacologia , Exocitose/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Mucinas/metabolismo , Ácido Niflúmico/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cloretos/metabolismo , Capacitância Elétrica , Líquido Extracelular/metabolismo , Células HT29 , Humanos , Imidazóis/farmacologia , Mucosa Intestinal/metabolismo , Membranas Intracelulares/metabolismo , Lantânio/farmacologiaRESUMO
To test the hypothesis that Na+ transport in human bronchial epithelial (HBE) cells is regulated by a protease-mediated mechanism, we investigated the effects of BAY 39-9437, a recombinant Kunitz-type serine protease inhibitor, on amiloride-sensitive short-circuit current of normal [non-cystic fibrosis (CF) cells] and CF HBE cells. Mucosal treatment of non-CF and CF HBE cells with BAY 39-9437 decreased the short-circuit current, with a half-life of approximately 45 min. At 90 min, BAY 39-9437 (470 nM) reduced Na+ transport by approximately 70%. The inhibitory effect of BAY 39-9437 was concentration dependent, with a half-maximal inhibitory concentration of approximately 25 nM. Na+ transport was restored to control levels, with a half-life of approximately 15 min, on washout of BAY 39-9437. In addition, trypsin (1 microM) rapidly reversed the inhibitory effect of BAY 39-9437. These data indicate that Na+ transport in HBE cells is activated by a BAY 39-9437-inhibitable, endogenously expressed serine protease. BAY 39-9437 inhibition of this serine protease maybe of therapeutic potential for the treatment of Na+ hyperabsorption in CF.
Assuntos
Brônquios/metabolismo , Fibrose Cística/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/farmacologia , Sódio/metabolismo , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Valores de ReferênciaRESUMO
A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current (I(sc)), followed by a sustained inhibition of amiloride-sensitive I(sc). These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in I(sc) was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.
Assuntos
Brônquios/citologia , Células Epiteliais/metabolismo , Receptores de Trombina/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Expressão Gênica/fisiologia , Hemostáticos/farmacologia , Humanos , Oligopeptídeos/farmacologia , RNA Mensageiro/análise , Receptor PAR-2 , Receptores de Trombina/genética , Trombina/farmacologia , Tripsina/farmacologiaRESUMO
Leukocyte accumulation and activation are key events in the pathogenesis of inflammatory lung disease. The ability of human airway smooth muscle cells (HASM) to contribute to the inflammatory process by its ability to produce the chemokines interleukin (IL) 8, monocyte chemotactic protein (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) was investigated. Cultured HASM, when stimulated with the pro-inflammatory cytokines IL-1 alpha (0.01-1 ng/ml) or tumour necrosis factor alpha (TNF-alpha, 0.3-30 ng/ml), synthesize and release substantial amounts of IL-8, as assessed by specific immunoassay, bioasssay (elevation of intracellular free calcium in human neutrophils), and upregulation of mRNA. These stimuli also increased MCP-1 production and mRNA expression, but RANTES mRNA expression was not detected at 24 h. The smooth muscle spasmogen endothelin 1 (1 microM) was unable to stimulate IL-8 or MCP-1 release or mRNA expression. These data indicate that HASM may constitute an important source of leukocyte attractants in the inflamed lung, where the inducing stimuli, IL-1 alpha and TNF-alpha, are also likely to be present.
Assuntos
Brônquios/metabolismo , Quimiocina CCL2/biossíntese , Interleucina-8/biossíntese , Músculo Liso Vascular/metabolismo , Brônquios/citologia , Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL5/biossíntese , Endotelina-1/farmacologia , Expressão Gênica , Humanos , Interleucina-8/genética , Músculo Liso Vascular/citologia , Neutrófilos/metabolismo , RNA MensageiroRESUMO
The importance of phospholipase C catalysed hydrolysis of phosphatidylinositol-(4,5)bisphosphate (PtdIns(4,5)P2) to inositol-(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol in the signal transduction pathways of eukaryote cells, in response to extracellular stimuli, is now widely recognised. Although nearly 60 naturally occurring inositol phosphates have been identified in mammalian cells, mobilisation of Ca2+ from the intracellular stores has been most commonly attributed to the generation of Ins(1,4,5)P3 [1]. However, there is increasing evidence for the presence of ryanodine receptors (RyR) in non-excitable cells and for cADP-ribose (cADPr) as the signalling molecule responsible for Ca2+ release via the RyR. But what is the purpose for the co-existence of these two intracellular Ca2+ channels in non-excitable cells and why are they so heterogeneous in their distribution? These questions were explored at the recent International Symposium Calcium Signalling in Inflammatory Cells. Depletion of the intracellular Ca2+ pools is followed by entry of Ca2+ into the cell across the plasma membrane, but the mechanism(s) underlying this 'capacitative Ca2+ entry' is not well understood. Many potential signalling pathways which may account for capacitative Ca2+ entry have been proposed although none have been unanimously accepted. New developments in the elucidation of the mechanism responsible for capacitative Ca2+ entry and how Ca2+ entry is regulated, together with progress in the characterisation of plasma membrane Ca2+ entry channels were also discussed at this symposium.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
The contribution of receptor-operated Ca2+ channels (ROCs), voltage-operated Ca2+ channels (VOCs), and intracellular Ca2+ release to contractions induced by a range of stimuli in the guinea pig isolated trachea has been evaluated. In the presence of physiological Ca2+ (1.3 x 10(-3) M), tissue pretreatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (4 x 10(-3) M for 5 min) markedly inhibited (> 90%) the contractile responses to a range of agonists. Therefore, under physiological Ca2+ concentration, Ca2+ mobilization from internal stores appeared to make little contribution to maximum contractions. Nifedipine (10(-7) M) or verapamil (10(-5) M) abolished KCl-induced contractions but produced variable inhibition of contractions induced by other agonists. The ROC (and VOC) blocker, SK&F 96365 (10(-5)-10(-4) M), inhibited both KCl-induced contractions and the nifedipine-insensitive component of contractions induced by acetylcholine (ACh), U46619, or leukotriene D4 [half maximal inhibitory concentration (IC50) values 1.7-3.8 x 10(-5) M]. Ni2+, which has ROC- and VOC-blocking actions, also abolished nifedipine-insensitive contractions induced by ACh. When Ca2+ was replaced with Ba2+, the contraction induced by ACh was blocked by nifedipine. Also, under these conditions, ACh did not increase the KCl maximum contraction. These data are consistent with there being distinct ROC and VOC influx pathways in guinea pig trachea and with ROCs playing a significant role in smooth muscle contraction.
Assuntos
Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Contração Muscular/fisiologia , Traqueia/fisiologia , Animais , Bário/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Ácido Egtázico/farmacologia , Espaço Extracelular/metabolismo , Cobaias , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Níquel/farmacologia , Traqueia/metabolismoRESUMO
The relative potencies of PGD2, PGE2 and PGI2 in potentiating bradykinin-induced hyperalgesia and oedema were determined in the paws of aspirin-treated guinea-pigs. PGE2 and to a lesser degree PGD2 but not PGI2, potentiated bradykinin-induced hyperalgesia, whereas PGD2, but not PGE2 or PGI2, potentiated oedema. These findings differ from those in other species, and possibly reflect interspecies differences in modulation of inflammatory reactions by prostanoids.
Assuntos
Bradicinina/farmacologia , Edema/prevenção & controle , Dor/prevenção & controle , Prostaglandinas/farmacologia , Animais , Aspirina/farmacologia , Dinoprostona/farmacologia , Edema/induzido quimicamente , Epoprostenol/farmacologia , Feminino , Cobaias , Dor/induzido quimicamente , Prostaglandina D2/farmacologia , Limiar Sensorial/efeitos dos fármacosRESUMO
A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.