Date of Award
Master of Science (MS)
Mitochondrial membrane potential (Δm) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction in various cell types. The objective of this study was to develop an experimental and computational approach for estimating Δm in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were isolated and connected to a ventilation-perfusion system. The experimental Protocol consisted of three single-pass phases: loading, wash, and uncoupling, in which the lungs were perfused with R6G-containing perfusate, fresh R6G-free perfusate, or R6G-free perfusate containing the mitochondrial uncoupler FCCP, respectively. This Protocol was carried out with or without lung perfusate containing verapamil, an inhibitor of the multi-drug efflux pump P-glycoprotein.Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration, and that this increase was larger in the presence of verapamil than in its absence. A physiologically based pharmacokinetic (PBPK) model for the pulmonary disposition of R6G was developed and used for quantitative interpretation of the kinetic data, including estimating Δm. The estimated value of Δm (-139 ± 21 (SD) mV and -128 ± 14 mV without and with verapamil, respectively) is consistent with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying Δm in intact functioning lungs. This approach has potential to provide quantitative assessment of the effect of injurious conditions on lung mitochondrial function, and to evaluate the impact of therapies that target mitochondria.