RESUMEN
The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
Asunto(s)
Antiasmáticos/farmacología , Canales de Cloruro/metabolismo , Palmitatos/farmacología , Canales de Potasio Calcio-Activados/metabolismo , Receptor PAR-2/agonistas , Animales , Señalización del Calcio , Células Cultivadas , Evaluación Preclínica de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Activación del Canal Iónico , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Ornitina/análogos & derivados , Ornitina/farmacología , Receptor PAR-2/metabolismo , Mucosa Respiratoria/citología , Tráquea/citologíaRESUMEN
Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic arsenic and metabolites were significantly higher in Ajo (20.6±3.5 µg/l, 0.54±0.77 µg/g and 27.7±21.2 µg/l, respectively) than in Tucson (3.9±2.5 µg/l, 0.16±0.20 µg/g and 13.0±13.8 µg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium positively associated with sputum AAT (P=0.004 and P=0.002, respectively). In analyses stratified by town, these relationships remained significant only in Ajo, with the higher arsenic exposure. Reduction in AAT may be a means by which arsenic induces respiratory disease, and selenium may protect against this adverse effect.
Asunto(s)
Arsénico/toxicidad , Exposición a Riesgos Ambientales , Selenio/farmacología , Esputo/metabolismo , Contaminantes Químicos del Agua/toxicidad , alfa 1-Antitripsina/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Paracrine ATP signaling in the lung epithelium participates in a variety of innate immune functions, including mucociliary clearance, bactericide production, and as an initiating signal in wound repair. We evaluated the effects of chronic low-dose arsenic relevant to U.S. drinking water standards (i.e., 10 ppb [130nM]) on airway epithelial cells. Immortalized human bronchial epithelial cells (16HBE14o-) were exposed to 0, 130, or 330nM arsenic (as Na-arsenite) for 4-5 weeks and examined for wound repair efficiency and ATP-mediated Ca(2+) signaling. We found that chronic arsenic exposure at these low doses slows wound repair and reduces ATP-mediated Ca(2+) signaling. We further show that arsenic compromises ATP-mediated Ca(2+) signaling by altering both Ca(2+) release from intracellular stores (via metabotropic P2Y receptors) and Ca(2+) influx mechanisms (via ionotropic P2X receptors). To better model the effects of arsenic on ATP-mediated Ca(2+) signaling under conditions of natural exposure, we cultured tracheal epithelial cells obtained from mice exposed to control or 50 ppb Na-arsenite supplemented drinking water for 4 weeks. Tracheal epithelial cells from arsenic-exposed mice displayed reduced ATP-mediated Ca(2+) signaling dynamics similar to our in vitro chronic exposure. Our findings demonstrate that chronic arsenic exposure at levels that are commonly found in drinking water (i.e., 10-50 ppb) alters cellular mechanisms critical to airway innate immunity.
Asunto(s)
Arsénico/toxicidad , Bronquios/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Bronquios/citología , Bronquios/metabolismo , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismoRESUMEN
Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Europio/química , Oligopéptidos/química , Oligopéptidos/farmacología , Receptor PAR-2/agonistas , Animales , Unión Competitiva , Línea Celular , Humanos , Oligopéptidos/síntesis química , Ácido Pentético/química , Ratas , Receptor PAR-2/metabolismo , Espectrometría de Fluorescencia , Coloración y Etiquetado , Factores de TiempoRESUMEN
Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.