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Quantification of Mitochondrial Membrane Potential in the Isolated Rat Lung Using Rhodamine 6G
Author(s) -
Audi Said H.,
Cammarata Anthony,
Clough Anne V.,
Dash Ranjan K.,
Jacobs Elizabeth R.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02263
Subject(s) - rhodamine 123 , verapamil , membrane potential , chemistry , mitochondrion , perfusion , lung , pharmacology , paraquat , biophysics , biochemistry , biology , medicine , calcium , organic chemistry , multiple drug resistance , antibiotics
Mitochondrial membrane potential ( ΔΨ m ) 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 ΔΨ m 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 ΔΨ m . The estimated value of ΔΨ m (−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 ΔΨ m 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 Information Supported 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.