Quantification of Mitochondrial Membrane Potential in the Isolated Rat Lung Using Rhodamine 6G

Abstract

Mitochondrial membrane potential (ΔΨm) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction. The objective of this study was to develop an experimental and computational approach for estimating ΔΨm in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were excised 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 lung perfusate containing verapamil vehicle or verapamil, an inhibitor of the multi‐drug efflux pump P‐glycoprotein (Pgp). Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration (Figure 1), 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 ΔΨm. The estimated value of ΔΨm (−139 ± 21 (SD) mV) was not significantly altered by inhibiting Pgp with verapamil, and is consistent with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying ΔΨm 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.Support or Funding InformationSupported by NIH 2R15HL129209‐02 and VA Merit Review Award BX001681.Solid symbols: R6G venous effluent concentrations during the loading phase, wash phase, and uncoupling phase using FCCP (67 μM). Values are mean ± SE (n = 7 for loading and wash phases, and n = 5 for uncoupling phase). Solid line is PBPK model fit to data.Figure 1