Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes. Am J Physiol Lung Cell Mol Physiol 2011 May;300(5):L762-72
Date
01/18/2011Pubmed ID
21239539Pubmed Central ID
PMC3094023DOI
10.1152/ajplung.00334.2010Scopus ID
2-s2.0-79955777883 (requires institutional sign-in at Scopus site) 25 CitationsAbstract
Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.
Author List
Gan Z, Audi SH, Bongard RD, Gauthier KM, Merker MPAuthor
Said Audi PhD Professor in the Biomedical Engineering department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
AnimalsCattle
Cells, Cultured
Endothelial Cells
Fluorescent Dyes
Hyperoxia
Membrane Potentials
Mitochondria
Organometallic Compounds
Pulmonary Artery
Rhodamine 123
