Scott A Boitano

The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections. Dye coupling studies revealed functional gap junctions between and among each cell phenotype. Localized mechanical stimulation resulted in propagated changes of intracellular Ca2+ to AT1 or AT2 cells independent of the stimulated cell phenotype. Ca2+ communication that originated after AT1 cell stimulation was inhibited by gap junction blockers, but not by an inhibitor of extracellular nucleotide signaling (apyrase). Conversely, Ca2+ communication after stimulation of AT2 cells was not significantly reduced by gap junction inhibitors. However, apyrase significantly reduced Ca2+ communication from AT2 to AT1 cells, but not from AT2 to AT2 cells. In conclusion, AT1 and AT2 cells have unique connexin profiles that allow for functional coupling and distinct intercellular pathways for coordination of Ca2+ signaling.

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.

Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.

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.

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.