Enhanced selectivity screening of GPCR ligands using a label-free cell based assay technology.

Drug discovery efforts advance in step with advancements in assay technologies, as new technologies provide new lenses through which biology can be viewed. The novel information gathered results in the better understanding of drug-target interactions leading to better decision making during the drug discovery process. One area of rapid development is within label-free technologies. Label-free technologies offer many distinct advantages to the drug discovery workflow. One such novel technology is the CellKey System, an impedance-based label-free live cell assay platform. The system is based on impedance technology and is a universal platform for the functional measurement of all classes of G-protein coupled receptors (GPCRs). Data are generated in a kinetic fashion on both endogenously expressed and transfected receptors in a wide variety of cell types. In the studies detailed here, we used the system to perform an enhanced selectivity screen of a small panel of compounds simultaneously against two unrelated GPCR targets signaling through different pathways. Utilizing both the quantitative measures of cellular activation and the qualitative information inherent in the rich output data, we gained knowledge not only about the relative selectivity of each compound across both targets, but also about the character of the interaction of each with the cellular target. In this manner, we successfully demonstrated proof of principal for using an impedance-based technology to perform selectivity analyses and to triage lead compounds in a simplified format.

Effect of dexamethasone on antigen-induced high molecular weight glycoconjugate secretion in allergic guinea pigs.

The ovalbumin-sensitized guinea pig is commonly used as a small animal model of allergic asthma. This animal model exhibits many of the hallmark characteristics observed in patients afflicted with asthma including nonspecific airway hyperreactivity, airway eosinophilia, early and late phase bronchoconstriction, and plasma extravasation into the airways. In addition, mucous hypersecretion in the airways of asthmatic patients is thought to be responsible for the plugging of distal airways and to contribute to the morbidity and mortality associated with the disease process. In this study we examined whether the allergic guinea pig model exhibits an increase in airway high molecular weight glycoconjugate (HMWG) secretion in response to an antigen challenge and whether dexamethasone exerts any modulatory effects upon the response. Ovalbumin (OVA) -sensitized guinea pigs were challenged with OVA 2 wk following the initial exposure. Trachobronchoalveolar lavages (TBAL) were performed, and the samples were assayed for total eosinophil cell number, eosinophil peroxidase activity (EPO), and both acidic and neutral HMWG content. Morphometric analysis of mucous-containing cells was also performed on tissue sections prepared from the trachea, mainstem bronchus, and three lobes of the left lung. Within 24 h of an antigen challenge, TBAL samples obtained from the allergic guinea pigs exhibited increases in eosinophil cell number, measured EPO enzyme activity, and acidic HMWG content compared to TBAL samples prepared from vehicle-exposed animals. These antigen-induced changes were dependent on the concentration of aerosolized OVA administered. Exposing the animals to 0.3% OVA provoked a 6.23-fold increase in airway eosinophils, 15-fold elevation in TBAL EPO enzyme activity, and 175% increase in TBAL acidic HMWG. No significant changes in TBAL neutral HMWG were measured. The changes in measured EPO activity correlated with the levels of acidic HMWG found in the TBAL samples (r = 0.73, P < or = 0.001). The measured increase in TBAL acidic HMWG was time dependent and was found to be maximal at 2 h post-antigen challenge. Morphometric analysis of Alcian blue (pH 2.5) -stained airway sections showed a decline in stored mucosubstances following the antigen exposure, supporting the notion that the allergic guinea pig model exhibits a mucosecretory component. Pretreating the animals with dexamethasone attenuated the antigen-induced release of HMWG and changes in measured EPO activity. In conclusion, these data indicate that the allergic guinea pig may be a useful model for examining the neural and cellular mechanisms underlying mucus hypersecretion in individuals afflicted with bronchial asthma.

Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis.

Chronic exposure of rats to high concentrations of SO2 gas causes pathologic changes in airway similar to those seen in human chronic bronchitis. The purpose of this study was to examine the pulmonary mechanical correlates of these changes and to quantify the extent of mucous hypersecretion by measuring changes in mucous glycoproteins. Female Sprague-Dawley rats were exposed to 250 ppm SO2 gas, 5 h/d, 5 d/wk, for a period of 4 wk. Control rats were exposed to air only. On the day after the last SO2 exposure, rats were anesthetized, instrumented for the measurement of pulmonary resistance (RL) and dynamic compliance (Cdyn), and ventilated. Chronic SO2 exposure caused a small but significant increase in RL and decrease in Cdyn. Airway responsiveness to inhaled aerosolized methacholine was increased in SO2-exposed rats, as indicated by approximately 6.6- and 4.6-fold decreases respectively, in the doses of inhaled methacholine required to double RL or decrease Cdyn to 50% of baseline. SO2 exposure had no effect on the contractile response of the trachea measured in vitro. Tracheae and lungs from SO2-exposed animals exhibited 140 and 535% increases in measured neutral mucous glycoproteins, respectively, and 33 and 37% increases in acid glycoproteins. Our results indicate that this animal model of chronic bronchitis mimics the mucous hypersecretion, airway obstruction, and increased airway responsiveness observed in human bronchitis and may allow us to begin to probe their mechanistic basis.

Hyperplastic effects of aerosolized sodium metabisulfite on rat airway mucus-secretory epithelial cells.

The ability of aerosolized sodium metabisulfite to induce hypertrophic and hyperplastic changes in rat airway secretory epithelial cells was investigated. A 10% solution of sodium metabisulfite was aerosolized into a Plexiglas exposure chamber, using an ultrasonic humidifier. The level of SO2 gas generated by this apparatus was measured to be 500 ppm. Measured levels of neutral and acidic mucous glycoproteins in extracts from tracheal and lung tissue were used as indices of hypertrophic (increases in mucus content per cell) and hyperplastic (increased numbers of cells containing mucus per gram of tissue) changes occurring in mucus-secreting cells of the airways. Exposing rats to sodium metabisulfite for 3 weeks resulted in profound increases in total neutral mucous glycoproteins found in tracheal and lung tissue (6.2-fold and 10.1-fold, respectively), compared with the H2O-treated counterparts. Total acidic mucous glycoproteins were significantly elevated in lung tissue only (13.5-fold). In addition, neutral and acidic mucous glycoproteins were elevated 20-fold and 9-fold, respectively, in bronchoalveolar lavage samples prepared from sodium metabisulfite exposed animals. These results indicate that aerosolized sodium metabisulfite may be a useful agent for developing small animal models of mucus hypersecretion.

Hypertrophic and hyperplastic changes of mucus-secreting epithelial cells in rat airways: assessment using a novel, rapid, and simple technique.

Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.

A perfused-cuvette method for fluorimetric studies of non-adherent cells.

We have developed a system for immobilizing non-adherent cells for use in macroscopic fluorescence measurements in a perfused cuvette. Using normal human T lymphocytes loaded with the fluorescent Ca(2+)-indicator, fluo-3, we have validated the properties of these cells immobilized on clear, non-fluorescent fluorohalocarbon film (Aclar) using the non-charged cell adhesive, Cell-Tak.