Quantifying Mitochondrial and Plasma Membrane Potentials in Intact Pulmonary Arterial Endothelial Cells Based on Extracellular Disposition of Rhodamine Dyes

Authors

Zhuohui Gan, Marquette University
Said H. Audi, Marquette UniversityFollow
Robert D. Bongard, Medical College of Wisconsin
Kathryn M. Gauthier, Medical College of Wisconsin
Marilyn P. Merker, Medical College of Wisconsin

Document Type

Article

Language

eng

Format of Original

11 p.

Publication Date

2011

Publisher

American Physiological Society

Source Publication

American Journal of Physiology: Lung Cellular and Molecular Physiology

Source ISSN

1040-0605

Original Item ID

doi: 10.1152/ajplung.00334.2010

Abstract

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% O₂ 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.

Comments

Accepted version. American Journal of Physiology: Lung Cellular and Molecular Physiology, Vol. 300, No. 5 (2011): L762-L772. DOI. © 2011 American Physiological Society. Used with permission.

Recommended Citation

Gan, Zhuohui; Audi, Said H.; Bongard, Robert D.; Gauthier, Kathryn M.; and Merker, Marilyn P., "Quantifying Mitochondrial and Plasma Membrane Potentials in Intact Pulmonary Arterial Endothelial Cells Based on Extracellular Disposition of Rhodamine Dyes" (2011). Biomedical Engineering Faculty Research and Publications. 256.
https://epublications.marquette.edu/bioengin_fac/256