Quantification of mitochondrial membrane potential in the isolated rat lung using rhodamine 6G

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, washing, 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 multidrug efflux pump P-glycoprotein (Pgp). 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 Δψm. The estimated value of Δψm [−144 ± 24 (SD) mV] was not significantly altered by inhibiting Pgp with verapamil and is comparable 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. NEW & NOTEWORTHY A novel experimental and computational approach for estimating mitochondrial membrane potential (Δψm) in intact functioning lungs is presented. The isolated rat lung inlet-outlet concentrations of the fluorescent cationic dye rhodamine 6G were measured and analyzed by using a computational model of its pulmonary disposition to determine Δψm. The approach has the potential to provide quantitative assessment of the effect of injurious conditions and their therapies on lung mitochondrial function.

Surface tension in situ in flooded alveolus unaltered by albumin

In the acute respiratory distress syndrome, plasma proteins in alveolar edema liquid are thought to inactivate lung surfactant and raise surface tension, T. However, plasma protein-surfactant interaction has been assessed only in vitro, during unphysiologically large surface area compression (%Δ A). Here, we investigate whether plasma proteins raise T in situ in the isolated rat lung under physiologic conditions. We flood alveoli with liquid that omits/includes plasma proteins. We ventilate the lung between transpulmonary pressures of 5 and 15 cmH2O to apply a near-maximal physiologic %Δ A, comparable to that of severe mechanical ventilation, or between 1 and 30 cmH2O, to apply a supraphysiologic %Δ A. We pause ventilation for 20 min and determine T at the meniscus that is present at the flooded alveolar mouth. We determine alveolar air pressure at the trachea, alveolar liquid phase pressure by servo-nulling pressure measurement, and meniscus radius by confocal microscopy, and we calculate T according to the Laplace relation. Over 60 ventilation cycles, application of maximal physiologic %Δ A to alveoli flooded with 4.6% albumin solution does not alter T; supraphysiologic %Δ A raise T, transiently, by 51 ± 4%. In separate experiments, we find that addition of exogenous surfactant to the alveolar liquid can, with two cycles of maximal physiologic %Δ A, reduce T by 29 ± 11% despite the presence of albumin. We interpret that supraphysiologic %Δ A likely collapses the interfacial surfactant monolayer, allowing albumin to raise T. With maximal physiologic %Δ A, the monolayer likely remains intact such that albumin, blocked from the interface, cannot interfere with native or exogenous surfactant activity.

Hypoxia recruits intrapulmonary arteriovenous pathways in intact rats but not isolated rat lungs

Intrapulmonary arteriovenous anastomoses (IPAVS) directly connect the arterial and venous circulations in the lung, bypassing the capillary network. Here, we used solid, latex microspheres and isolated rat lung and intact, spontaneously breathing rat models to test the hypothesis that IPAVS are recruited by alveolar hypoxia. We found that hypoxia recruits IPAVS in the intact rat, but not the isolated lung. IPAVS are at least 70 μm in the rat and, interestingly, appear to be recruited when the mixed venous Po2 falls below 22 mmHg. These data provide evidence that large-diameter, direct arteriovenous connections exist in the lung and are recruitable by hypoxia in the intact animal.