Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Abstract

The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.

Development of an Improved Tracheal Explant Bioassay for the Detection of the Ciliary Dyskinesia Factor in Cystic Fibrosis Serum

“…In an attempt to develop an improved bioassay for the CF factor, we utilized tracheal ring explants in which ciliary motion (observed along the inner, vertical surface of a 1-to 2-mm tracheal slice) could be quantitated visually. This culture system has been used with various biochemical (16,18,19) assays of viability, so there are standardized subjective and objective techniques available to measure necrosis. High concentrations of human serum (e.g., 50%) on explants caused little differential response in ciliary activity between control serum and CF serum, and no cytonecrosis was detectable.…”

“…Although significant improvements toward standardization have been instituted (4, l l , 12), the manipulation and scoring of the mucosal sheets remain cumbersome and are probably the source of many reported discrepancies. Tracheal ring organ, or explant, cultures have been used extensively in infectious diseases (8,15) and have advantages in ease of preparation and quantitation (15,18,19). We employed the explant model system with hamster, rabbit, and guinea pig tissue in an attempt to: 1) evaluate the utility of tracheal slices for detecting CF-related alterations in ciliary activity, and 2) biochemically assess the extent of cytotoxicity in CF-treated organ cultures.…”

Cytotoxicity and Ciliostasis in Tracheal Explants Exposed to Cadmium Salts

“…A prolonged lack of available ATP would also decrease cellular protein and nucleic acid synthesis and reduce the efficiency of the ion pumps necessary to maintain normal intracellular osmotic and ionic pressures (14,30). Our study supports this hypothesis, since it is possible that the measurable ATP losses which we found were a result of all death and thinning of the epithelium after the inactivation of ATPase.…”

“…RCA is the product of the percent of the surface with an intact epithelium (0-100%), and a subjective estimate of vigor of ciliary motion (0-3 +). Biochemical assays of cell viability were based on dehydrogenase activity (primarily succinate, as determined with a tetrazolium chloride reduction assay) (13) and ATP content (determined photometrically with a luciferin-luciferase enzyme system) (14). For histopathological examination, tracheal explants were rinsed in phosphate buffered saline (PBS, pH 7.4) and fixed in 4% glutaraldehyde in PBS.…”

Antimycoplasmal activity of dimethylphenols in a tracheal explant culture system

“…The events which follow attachment are currently unknown but may include the fusion of the host cell and mycoplasma membranes and the passage of metabolically active substances from the mycoplasmas into the host cell (23). At least one consequence of these events is the eventual interruption of the adenine-dependent energy metabolism of the host cell (7,11). Assuming that 2,4-DMP interferes with one or another of these sequentially occurring processes, the point at which this compound is added to the organ culture media may affect its ability to prevent infection.…”