Marilyn P. Merker

Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells

Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.

Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia

The objective was to evaluate the pulmonary disposition of the ubiquinone homolog coenzyme Q(1) (CoQ(1)) on passage through lungs of normoxic (exposed to room air) and hyperoxic (exposed to 85% O(2) for 48 h) rats. CoQ(1) or its hydroquinone (CoQ(1)H(2)) was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of CoQ(1)H(2) and CoQ(1) were measured. CoQ(1)H(2) appeared in the venous effluent when CoQ(1) was infused, and CoQ(1) appeared when CoQ(1)H(2) was infused. In normoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 58 and 33% in the presence of rotenone (mitochondrial complex I inhibitor) and dicumarol [NAD(P)H-quinone oxidoreductase 1 (NQO1) inhibitor], respectively. Inhibitor studies also revealed that lung CoQ(1)H(2) oxidation was via mitochondrial complex III. In hyperoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 23% compared with normoxic lungs. Based on inhibitor effects and a kinetic model, the effect of hyperoxia could be attributed predominantly to 47% decrease in the capacity of complex I-mediated CoQ(1) reduction, with no change in the other redox processes. Complex I activity in lung homogenates was also lower for hyperoxic than for normoxic lungs. These studies reveal that lung complexes I and III and NQO1 play a dominant role in determining the vascular concentration and redox status of CoQ(1) during passage through the pulmonary circulation, and that exposure to hyperoxia decreases the overall capacity of the lung to reduce CoQ(1) to CoQ(1)H(2) due to a depression in complex I activity.

Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes.

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.

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells.

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.

Cyanide increases reduction but decreases sequestration of methylene blue by endothelial cells

AbstractThe mechanisms of endothelial cell transplasma membrane electron transport (TMET) have not been completely identified. Redox probes such as methylene blue (MB) can be useful tools, but the complexity of their disposition upon exposure to the cells can hinder interpretation. For example, MB is reduced on the cell surface by TMET, but after entering the cell in reduced form, it is reoxidized and sequestered within the cell. We developed a method to separately quantify the reduction and reoxidation rates such that it can be determined whether a metabolic inhibitor such as cyanide affects the reduction or oxidation process. MB was introduced at the inlet to a column filled with endothelial cell covered beads either as a short 12 s injection (bolus) or a long 45 min infusion (pulse), and its effluent concentration was measured as a function of time. The cells extracted 56% of the MB from the bolus, but only 41% during the pulse steady state. In the presence of cyanide, these extractions increased to 70% and decreased to 4%, respectively. Mathematical model results support the interpretation that these paradoxical effects on bolus and pulse extractions reflect the differential effects of cyanide on extracellular reduction and intracellular oxidation, i.e., cyanide increased the reduction rate from 7.3 to 13.0 cm s-1 x 10-5 and decreased the oxidation rate from 1.09 to 0.02 cm s-1 x 10-3.Cyanide also increased intracellular NADH by almost eight times, suggesting that TMET is sensitive to the cell redox status, i.e., NADH is a direct or indirect electron source. The cyanide-induced decrease in sequestration indicates a cyanide-sensitive intracellular oxidation mechanism. The results also demonstrate the potential utility of this approach for further evaluation of these endothelial redox mechanisms.

Cyclophilin-facilitated bradykinin inactivation in the perfused rat lung

Cis and trans isomers of X-proline (X-Pro) bonds can influence some aspects of the kinetics of peptide metabolism. We previously used the peptidyl-prolyl cis-trans isomerase, cyclophilin, to show that angiotensin converting enzyme (ACE) preferentially hydrolyzes the trans isomer of a synthetic tripeptide that contains a C-terminal proline (Dawson et al., Am J Physiol 257:H853-H865, 1989; Merker et al., J Appl Physiol 75: 1519-1524, 1993). Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) exists as both cis and trans isomers at all three X-Pro bonds, and although its inactivation in the lung by pulmonary endothelial peptidases is extensive, commonly a small fraction of the peptide survives passage through the lung. To determine whether the presence of cis X-Pro bonds might limit the extent of bradykinin metabolism in the lung, we studied inactivation of bradykinin by the isolated perfused rat lung using the rabbit jugular vein superfused with the pulmonary venous effluent as a bioassay for bradykinin. A large fraction (> 90%) of the bradykinin in a bolus injection was inactivated in a single transit through the pulmonary circulation, but a detectable fraction emerged in the venous effluent. The addition of cyclophilin to the bradykinin in the bolus reduced the bradykinin emerging from the lungs to virtually undetectable levels. When the isomerase inhibitor cyclosporin A was included with bradykinin and cyclophilin in the injectate, this effect of cyclophilin was reversed. These observations suggest that the fraction of bradykinin that normally survives passage through the lungs contains isomers that have at least one X-Pro bond that is refractory to enzymatic inactivation and whose isomerization time constant is significantly longer than the pulmonary capillary transit time.

Characterization of the threshold for NAD(P)H:quinone oxidoreductase activity in intact sulforaphane-treated pulmonary arterial endothelial cells.

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5μM, 24h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP(+) in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP(+) ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K(m) for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold.

Kinetics of Plasma Membrane Electron Transport in a Pulmonary Endothelial Cell-Column

Thiazine dyes such as toluidine blue O (TBO) are reduced at the luminal endothelial surface. The purpose of this study was to determine the rate of this reaction in endothelial cells in culture. A multiple indicator dilution method was used to measure the reaction kinetics during transient passage of a TBO-containing bolus through a chromatographic column filled with bovine pulmonary arterial endothelial cells grown on microcarrier beads (cell-column). A bolus containing TBO and an inert extracellular reference indicator (FITC-Dextran) was injected upstream of the cell-column, and the indicator concentrations were measured downstream using on-line photodetection. The effects of column flow rate, PO2, and TBO concentration were studied. The fraction of TBO reduced upon passage through the cell-column decreased with increasing flow indicating that the reaction rate rather than TBO delivery controlled TBO reduction. The fraction of TBO reduced did not change with PO2 or dose in the ranges studied. TBO reduction was about 10 times that for steady state TBO sequestration by these cells which, along with the lack of a PO2 effect indicates that the rapid rate of reduction is not the rate-limiting step in steady state sequestration.PAC98: 8722Fy, 8220-w

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1).

In isolated rat lung perfused with a physiological saline solution (5.5mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (Kf), effects that are overcome using an amphipathic quinone (CoQ1) [Free Radic. Biol. Med.65:1455-1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8nmol/mg protein, depolarized the mitochondrial membrane potential (Δψm) from -129.0±3.7 (mean±SEM) to -92.8±5.5mV, and decreased O2 consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC-dextran (~40kDa) continually over a 6 h time course. When CoQ1 was present with rotenone, normal ATP (17.4±1.4nmol/mg protein), O2 consumption (1.5±0.1nmol/min/mg protein), Δψm (-125.2±3.3mV), and permeability (PS) were maintained. Protective effects of CoQ1 on rotenone-induced changes in ATP, O2 consumption rate, Δψm, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol-mitochondria electron shuttle [Free Radic. Biol. Med.65:1455-1463; 2013]. Key rotenone effects without and with CoQ1 were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2mM.