Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

Elena N. Dedkova and Lothar A. Blatter

Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois

Submitted 12 January 2005 ; accepted in final form 16 May 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca2+ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca2+ uptake and Ca2+ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca2+ levels ([Ca2+]mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca2+ uptake, whereas inhibition of mtNOS with 10 µM L-N5-(1-iminoethyl)ornithine resulted in a significant increase of [Ca2+]mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca2+-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca2+ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca2+ overload under pathological conditions where cellular [NO] can reach very high levels.

nitric oxide synthase; permeability transition pore; endothelium


IN THE VASCULAR ENDOTHELIUM, nitric oxide (NO) plays an important regulatory role. NO, which is synthesized in a Ca2+-dependent manner by the endothelial NO synthase (eNOS), acts on vascular smooth muscle cells in the vessel wall as an endothelium-derived relaxing factor (40). We have demonstrated previously (19) that in vascular endothelial cells NO synthesis is not only Ca2+ dependent but also under an autoregulatory control that involves NO-dependent regulation of cytoplasmic Ca2+. Considering the known inhibitory effects of NO on cell respiration (6, 7, 18) and the recent discovery of a mitochondrial NOS (mtNOS) in endothelial cells (20), the question arises whether a similar autoregulatory function of NO in endothelial cells also affects mitochondrial Ca2+ uptake.

Although mtNOS has been discovered in a variety of cell types (3, 4, 20, 21, 25, 26, 35, 36, 38, 49, 52), the functional implications of NO produced locally by mitochondria for mitochondrial energy metabolism and Ca2+ homeostasis are much less explored and not well understood. NO is known to be a mediator of Ca2+ homeostasis in a highly complex and cell-specific manner (17), which can affect mitochondrial Ca2+ homeostasis as well. Several studies have addressed the effect of NO on mitochondrial Ca2+ homeostasis; however, the results are inconsistent and appear to depend on the cell types and experimental approaches (intact cells or isolated mitochondria) as well as the sources and the concentrations of NO used. For example, local suppression of mitochondrial Ca2+ handling by NO was proposed to be a key mechanism in the regulation of capacitative Ca2+ entry in human embryonic kidney cells (50), whereas no role for mitochondria in the regulation of capacitative Ca2+ entry by NO was found in platelets (51) and vascular endothelial cells (19). The experiments performed on isolated mitochondria have shown that application of NO donors inhibited Ca2+ uptake by mitochondria (9) or induced Ca2+ efflux from Ca2+-loaded mitochondria (2, 45), whereas blocking of mtNOS increased Ca2+ buffering capacity of mitochondria (27). These effects of NO were explained either by the ability of NO to decrease mitochondrial membrane potential ({Delta}{Psi}m) (2, 9, 27) or by the ability of NO to open the mitochondrial permeability transition pore (PTP) (9). It has also been suggested that the effects of NO on the PTP were not mediated by NO itself but rather by other reactive nitrogen species such as peroxynitrite (ONOO) (27, 48). It is noteworthy that in experiments performed on isolated mitochondria, higher concentrations of NO donors were used compared with experiments in intact cells, which could explain the difference in the results.

Bearing in mind the inconsistencies of previous reports on the effect of NO on mitochondrial Ca2+ handling, the present study was designed to examine the effect of a wide range of NO concentrations on mitochondrial Ca2+ uptake, {Delta}{Psi}m, and activity of the PTP in permeabilized calf pulmonary artery endothelial (CPAE) cells. The results of our study show that NO provided by the exogenous NO donor Sper/NO induced a dose-dependent inhibition of mitochondrial Ca2+ uptake and reduction of Ca2+ extrusion rates, but only when higher concentrations (100–500 µM) were used. Lower concentrations of Sper/NO (<100 µM) did not affect mitochondrial Ca2+ uptake; stimulation of mitochondrial NO production by supplying extra amounts of the mtNOS substrate L-arginine also resulted in an impairment of mitochondrial Ca2+ uptake, whereas abolishing mitochondrial NO production by blocking mtNOS with L-NIO resulted in an increase of mitochondrial Ca2+ uptake. NO induced a dose-dependent depolarization of the mitochondrial membrane, whereas L-NIO partially prevented the drop in membrane potential during mitochondrial Ca2+ uptake. Finally, higher concentrations of Sper/NO (100–500 µM) induced the opening of the mitochondrial PTP, which serves as additional mechanism for Ca2+ efflux from mitochondria. The results indicate that in CPAE cells, mitochondrially produced NO plays a protective role against mitochondrial Ca2+ overload through a negative feedback regulation of its own synthesis.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
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Cell Culture and Solutions

Experiments were performed on CPAE cells in nonconfluent cultures. The CPAE cell line was obtained at passage 15 from American Type Culture Collection (ATCC CCL-209, Manassas, VA). The cells were cultured in Eagle’s minimum essential medium, supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY) and 2 mM L-glutamine, and kept at 37°C in an atmosphere of 5% CO2 and 95% air. Once per week, the cells were dispersed with the use of a Ca2+-free (0.1% EDTA) 0.25% trypsin solution and subcultured onto glass coverslips for later experimentation. Cells were passaged up to 6 times after they were obtained from ATCC. All experiments were carried out at room temperature (20–22°C) on single cells in nonconfluent cultures within 1 wk after being plated.

Fluorescence Measurements

Mitochondrial Ca2+ measurements. Laser scanning confocal microscopy (model LSM 410, Zeiss) was used to follow the changes in the mitochondrial Ca2+ level ([Ca2+]mt) during activation of mitochondrial Ca2+ uptake. For fluorescence measurements, the coverslip with attached cells was mounted on the stage of an inverted microscope equipped with a x40 oil-immersion objective (Plan-Neofluar, 1.3 numerical aperture, Zeiss). Measurements of [Ca2+]mt were performed on cells loaded with the fluorescent Ca2+-sensitive dye fluo-3. Cells were exposed to 25 µM of the membrane-permeant form of the indicator fluo-3 AM (Molecular Probes, Eugene, OR) for 40 min at 37°C in 1 ml of standard Tyrode solution containing (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.3). Cells were subsequently washed for 10 min. The cells were then placed in Ca2+-free Tyrode solution and permeabilized by exposure to 10 µM digitonin for 60 s. Digitonin was added to the "intracellular" solution consisting of (in mM) 135 KCl, 10 NaCl, 20 HEPES, 5 pyruvate, 2 glutamate, 2 malate, 0.5 KH2PO4, 1 MgCl2, 5 EGTA, and 1.86 CaCl2 to yield a free [Ca2+] of ~100 nM. Fluo-3 fluorescence was excited with the 488-nm line of an argon ion laser, and the emitted fluorescence signals were measured at 510–525 nm. Changes in mitochondrial fluo-3 fluorescence intensities (F) in each experiment were normalized to the level of fluorescence recorded before stimulation (F0) but after cell permeabilization (see Fig. 1D). Changes in [Ca2+]mt are expressed as {Delta}F/F0. [Ca2+]mt measurements were performed from small mitochondria-rich regions of interest of ≤9 µm2, representing a small (<10) number of mitochondria.



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Fig. 1. Measurements of mitochondrial Ca2+ levels ([Ca2+]mt) in single permeabilized calf pulmonary artery endothelial (CPAE) cells with compartmentalized fluo-3. A fluo-3-loaded CPAE cell before (A) and after permeabilization with 10 µM digitonin (B) and subsequent increase of extramitochondrial [Ca2+] ([Ca2+]em) from 0.1 to 2 µM (C). Images were taken at the times indicated by the arrows in D. D: typical fluo-3 signals recorded from individual CPAE cells following digitonin addition and increase of [Ca2+]em in control conditions (red trace, control) and when mitochondrial Ca2+ uptake was blocked with 1 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; black trace) or 10 µM of ruthenium red (RutRed; blue trace). The fluorescence level after digitonin treatment (Fo) was used to normalize the fluo-3 signal (F/Fo) in all subsequent experiments. The rapid increase in fluo-3 fluorescence upon increasing [Ca2+] in the intracellular solution from 0.1 to 2 µM suggests Ca2+ uptake into the mitochondrial matrix.

 
Changes in {Delta}{Psi}m were followed using the potential-sensitive dye tetramethylrodamine ethyl ester (TMRE). CPAE cells were exposed to 0.2 µM TMRE for 15 min at 37°C before experiments and then permeabilized with digitonin. All solutions contained 0.2 µM TMRE during recordings. TMRE fluorescence was excited at 514 nm and recorded at 590 nm. For measurements of the time-dependent TMRE fluorescence changes, data were acquired every 2 s. Because the relationship between TMRE fluorescence and {Delta}{Psi}m is governed by the Nernst equation, TMRE fluorescence recordings are shown on a logarithmic scale.

Activity of mitochondrial permeability transition pore was monitored using the fluorescent dye calcein in permeabilized CPAE cells. Opening of PTP resulted in the loss of mitochondria-trapped calcein (620 kDa) and a decrease of fluorescence (34). CPAE cells were loaded with 5 µM of the membrane-permeant form of the fluorescent probe calcein AM (Molecular Probes) for 40 min at 37°C. After dye loading, the cells were placed in dye-free Tyrode solution for 10 min to wash off excess dye. The calcein fluorescence was excited at 488 nm, and the emitted fluorescence signal was measured at ~510 nm.

Chemicals

The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), ruthenium red (RutRed), decylubiquinone (DQ), and digitonin were obtained from Sigma (St. Louis, MO). Spermine NONOate (Sper/NO), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (PTIO), and L-N5-(1-iminoethyl)ornithine (L-NIO) were purchased from Calbiochem (San Diego, CA). Sper/NO was dissolved as a 15 mM stock in water before the experiments and used within 4 h.

Statistical Analysis

Statistical differences of the data were determined using Student's t-test for unpaired or paired data and were considered significant at P < 0.05. Results are reported as means ± SE for the indicated number (n) of cells. Each experiment was conducted on a separate cell culture.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of NO on Mitochondrial Ca2+ Uptake

For the direct measurement of Ca2+ uptake by mitochondria of vascular endothelial cells, we used a method based on the ability of the Ca2+-sensitive fluorescent indicator fluo-3 AM to compartmentalize into mitochondria, followed by subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. We have developed this method for CPAE cells and employed it successfully to estimate Ca2+ levels in mitochondria of endothelial cells (46). Figure 1A (before digitonin) shows that fluo-3 fluorescence was distributed relatively homogeneously throughout the cell with higher intensities of fluorescence around the nuclei. Plasma membrane permeabilization with digitonin removed cytosolic and nuclear fluo-3 (Fig. 1B, after digitonin), revealing the particulate and punctate fluorescence pattern typical for mitochondria (20). The mitochondrial origin of the fluo-3 signal was confirmed by colocalization with the potentiometric dye TMRE used to localize mitochondria (46). Elevating extramitochondrial [Ca2+] ([Ca2+]em) from 0.1 to 2 µM resulted in an increase of the mitochondria-trapped fluo-3 signal due to mitochondrial Ca2+ uptake (Fig. 1C). Figure 1D presents the typical time course of fluo-3 fluorescence intensity changes from mitochondria of CPAE cells before and after cell permeabilization with digitonin and after activation of mitochondrial Ca2+ uptake by increasing [Ca2+]em transiently from 0.1 to 2 µM (control, red trace). The level of fluorescence after digitonin treatment (F0) was on average only 21 ± 1% (n = 50) of the initial fluo-3 fluorescence. This level represents the contribution of mitochondria and was used to normalize the fluorescence signal (F/F0). When [Ca2+]em was raised from 0.1 to 2 µM, F/F0 increased to 5.81 ± 0.19 (measured at the peak of the response; n = 50). Figure 1D (black trace) shows the effect of dissipation of the {Delta}{Psi}m with the uncoupler of oxidative phosphorylation FCCP on mitochondrial Ca2+ accumulation. When cells were pretreated with 1 µM FCCP, elevation of [Ca2+]mt was virtually abolished (Fig. 1D, FCCP). Next we blocked the mitochondrial Ca2+ uniporter with 10 µM RutRed (Fig. 1D). After the cell was permeabilized with digitonin, 10 µM RutRed was added to the intracellular solution for 2 min before [Ca2+]em (blue trace) was increased. In the presence of RutRed, F/F0 increased to only 1.09 ± 0.02 (n = 8) in response to increasing [Ca2+]em from 0.1 µM up to 2 µM, while in the presence of 1 µM FCCP, the amplitude of F/F0 increased to 1.51 ± 0.08 (n = 9). In both instances the increases of F/F0 were significantly smaller (P < 0.001) than under control conditions. The rise in [Ca2+]mt amounted on average to only 2 and 11% of control in the presence RutRed and FCCP, respectively. Thus the increase of the fluo-3 signal after elevation of [Ca2+]em represents a Ca2+ uniporter-mediated, {Delta}{Psi}m-dependent Ca2+ uptake into the mitochondria.

After establishing the basic experimental protocol, we studied the effect of NO on mitochondrial Ca2+ uptake. Figure 2A shows a typical control recording of [Ca2+]mt from permeabilized CPAE cells in response to stimulation of mitochondrial Ca2+ uptake induced by increasing [Ca2+]em from 0.1 to 2 µM. After [Ca2+]em was switched back to the initial level (0.1 µM), mitochondrial Ca2+ also decreased quickly due to Ca2+ extrusion via mitochondrial Na+/Ca2+ exchange (46). The subsequent second increase of [Ca2+]em resulted in an identical [Ca2+]mt response (Fig. 2A, gray trace). This dual-application protocol was used in the following experiments to normalize the changes in [Ca2+]mt caused by Sper/NO (or other interventions) in individual cells. We used the NO donor Sper/NO in the range of 50–500 µM, which produces NO levels of 0.5–3 µM as estimated by us and reported by others (10, 13, 47). Therefore, the low concentrations of Sper/NO (50–100 µM) imitate NO production observed under physiological conditions (~0.5–1 µM NO), whereas higher concentrations of Sper/NO (>100 µM) produce NO in the range typical for pathological conditions (>1 µM NO) (24).



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Fig. 2. Modulation of [Ca2+]mt by nitric oxide (NO) provided by the exogenous NO donor spermine NONOate (Sper/NO). A: representative control traces of [Ca2+]mt changes (F/F0) in response to two subsequent applications of 2 µM Ca2+ to the same cell. B: shown are typical traces of fluo-3 fluorescence after addition of 2 µM Ca2+ in control conditions (black) and after the cell was pretreated with 300 µM Sper/NO for 2 min (gray). C: summary of normalized effects of Sper/NO on mitochondrial Ca2+ uptake and extrusion. The rate constant of Ca2+ extrusion (K = 1/{tau}) was calculated from a monoexponential fit to the decline of [Ca2+]mt after Ca2+ removal and was normalized to control values (%control). The numbers in parentheses indicate the number of cells tested. +P < 0.05; *P < 0.001, statistical significance compared with control values.

 
The application of the NO donor Sper/NO (300 µM), 2 min before elevation of [Ca2+]em, significantly decreased mitochondrial Ca2+ uptake (53 ± 3% of control, n = 15, P < 0.001; Fig. 2B, gray trace) and slowed Ca2+ extrusion after removal of extramitochondrial Ca2+ (Fig. 2B, gray trace). The rate of Ca2+ extrusion was calculated from a monoexponential fit to the decline of [Ca2+]mt during Ca2+ removal and normalized to control values (% of control). In the presence of 300 µM Sper/NO the rate of Ca2+ extrusion was reduced to 47 ± 6% of control (n = 15; P < 0.001). Low concentration of Sper/NO (50 µM) had no significant effect on mitochondrial Ca2+ uptake (94 ± 8% of control, n = 15; Fig. 2C) as well as on the rate of Ca2+ extrusion (102 ± 9% of control, n = 15; Fig. 2C). All data are summarized in Fig. 2C and indicate that the ability of mitochondria to sequester and extrude Ca2+ was significantly impaired when cells were treated with 100–500 µM Sper/NO. Lower concentrations of Sper/NO (<100 µM) had no effect. The close correlation between the degree of inhibition of mitochondrial Ca2+ uptake and the degree of inhibition of Ca2+ extrusion suggests that Ca2+ extrusion was slowed down as a result of the Sper/NO effect on mitochondrial Ca2+ uptake rather than a direct effect on Ca2+ extrusion mechanisms.

Effect of Stimulation and Inhibition of Mitochondrial NO Production by Constitutive mtNOS on Mitochondrial Ca2+ Uptake

In the above experiments, we used an exogenous NO source (NO donor Sper/NO) to evaluate the effect of NO on mitochondrial Ca2+ uptake. However, we have shown previously that stimulation of mitochondrial Ca2+ uptake activates mtNOS to produce NO inside of mitochondria of vascular endothelial cells, and with the experimental approach used here (permeabilized cells) the observed changes of [Ca2+]mt were exclusively due to mtNOS activity (20). For this reason, we set out to manipulate (increase or decrease) mitochondrial NO production to evaluate the effect of endogenously produced NO on [Ca2+]mt. To enhance mitochondrial NO production, we preincubated CPAE cells with the mtNOS substrate L-arginine. Despite the fact that endothelial cells can synthesize basal amounts of L-arginine, NO production is strongly dependent on the availability of exogenous L-arginine (30, 32). Therefore, we enhanced mitochondrial NO production by supplying an extra amount of L-arginine and evaluated how this affected mitochondrial Ca2+ uptake. Figure 3A presents a typical trace recorded from a cell pretreated with 1 mM L-arginine for 4 min before the second Ca2+ application. The experiment shows that stimulation of mitochondrial NO production resulted in an attenuation of mitochondrial Ca2+ uptake. The average increase of [Ca2+]mt during L-arginine treatment amounted to only 61 ± 3% of control (n = 19, P < 0.001; Fig. 3C).



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Fig. 3. Modulation of [Ca2+]mt by endogenous NO. A: representative traces of [Ca2+]mt changes (F/F0) in response to 2 µM Ca2+ in control conditions (left trace) and when cells were pretreated with 1 mM L-arginine for 4 min (right trace). B: representative traces of [Ca2+]mt changes in response to 2 µM Ca2+ in control conditions (left trace) and when cells were pretreated with 10 µM L-N5-(1-iminoethyl)ornithine (L-NIO) for 4 min before the second Ca2+ application (right trace). C: summary of the effects of eNOS substrate L-arginine and eNOS inhibitor L-NIO on mitochondrial Ca2+ uptake. The numbers in parentheses indicate the number of cells tested. *P < 0.001, statistically significant difference compared with control.

 
To decrease mitochondrial NO production, we blocked the activity of mtNOS with L-NIO, an inhibitor of the constitutive eNOS. Figure 3B shows that application of 10 µM L-NIO just 4 min before the second exposure to 2 µM Ca2+ enhanced mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake in the presence of L-NIO amounted to an increase of F/F0 to 128 ± 3% of control (n = 12, P < 0.001; Fig. 3C).

In summary, these data strongly suggest that NO can serve as a negative modulator of mitochondrial Ca2+ uptake and its own synthesis.

Effect of Sper/NO on {Delta}{Psi}m

To investigate mitochondrial function in permeabilized CPAE cells further, we used the potentiometric dye TMRE. Sequestration of TMRE into mitochondria is governed by the highly negative {Delta}{Psi}m, which is maintained by proton translocation by the electron transport system. When electron transport is inhibited pharmacologically or when the proton gradient is abolished by a protonophore, the membrane depolarizes, and TMRE is released into the cytoplasm, which leads to a decrease in TMRE fluorescence (Fig. 4A). We applied the protonophore FCCP (1 µM) at the end of each experiment. FCCP induced a complete depolarization of the mitochondrial membrane and a maximal decrease of TMRE fluorescence. We used this signal to normalize the changes in fluorescence produced by Sper/NO and other agents. TMRE exhibits a Nernstian distribution across the inner mitochondrial membrane; therefore, TMRE fluorescence signals are presented on a logarithmic scale. Sper/NO application caused a dose-dependent depolarization of {Delta}{Psi}m. Figure 4A shows a representative example of measurements of {Delta}{Psi}m-dependent TMRE fluorescence in response to application and withdrawal of 300 µM Sper/NO and subsequent application of 1 µM FCCP. Sper/NO produced a rapid drop in TMRE fluorescence that was stable during 2 min of Sper/NO exposure. The withdrawal of Sper/NO resulted in recovery of TMRE fluorescence to the initial level. While application of 300 µM Sper/NO had a profound effect on {Delta}{Psi}m, lower concentrations of Sper/NO had no or little effect (Fig. 4, B and F). Figure 4F summarizes the effect of different Sper/NO concentrations on the {Delta}{Psi}m. When cells were pretreated with 50 µM of the NO scavenger PTIO (1), Sper/NO-induced changes in TMRE fluorescence were abolished (11 ± 2% of the effect achieved with 300 µM Sper/NO alone, n = 9; P < 0.001; Fig. 4, C and F). Pretreatment with Mn-TBAP (50 µM), a cell-permeable superoxide dismutase (SOD) mimetic and ONOO scavenger, did not significantly change the effect of 300 µM Sper/NO on mitochondrial TMRE fluorescence (Fig. 4, D and F). In a total of 5 cells, Sper/NO-induced changes in TMRE fluorescence in the presence of Mn-TBAP were 116 ± 5% of the effect achieved with 300 µM Sper/NO alone (not significantly different; Fig. 4, D and F). The decrease in {Delta}{Psi}m induced by Sper/NO could be the result of the opening of the PTP, which renders the inner mitochondrial membrane permeable to molecules up to 1,500 Da (28, 53). The decrease in TMRE fluorescence induced by 300 µM Sper/NO was partially prevented by cell treatment with 100 µM of DQ (46 ± 2% of the effect achieved with 300 µM Sper/NO alone; n = 14, P < 0.001), a PTP inhibitor with a reported efficiency to block PTP comparable to that exerted by cyclosporin A (CsA) (23). We chose DQ over CsA because DQ has no effect on cell respiration (23) and CsA has been shown to be an unreliable blocker of PTP in CPAE and other cells (15, 33, 53).



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Fig. 4. Effect of NO on mitochondrial membrane potential ({Delta}{Psi}m). A: tetramethylrhodamine ethyl ester (TMRE) fluorescence changes during application and withdrawal of 300 µM Sper/NO and subsequent application and withdrawal of 1 µM FCCP. B: application 100 µM Sper/NO and 1 µM FCCP in control conditions. C: application of 300 µM Sper/NO and 1 µM FCCP in the presence of the NO scavenger PTIO (50 µM). D: application of 300 µM Sper/NO and 1 µM FCCP in the presence of the superoxide dismutase (SOD) mimetic and peroxynitrite scavenger Mn-TBAP (50 µM). E: application of 300 µM Sper/NO and 1 µM FCCP in the presence of the PTP inhibitor decylubiquinone (DQ; 100 µM). F: summary of the normalized {Delta}{Psi}m-dependent TMRE fluorescence changes upon application of 1 µM FCCP (100%) and different Sper/NO concentrations ([Sper/NO] in µM indicated by the numbers on the bottom of bars), 300 µM Sper/NO in the presence of 50 µM PTIO, 300 µM Sper/NO in the presence of 50 µM Mn-TBAP, 300 µM Sper/NO in the presence of 100 µM DQ. The fluorescence level after FCCP addition was used to normalize the Sper/NO-induced changes in TMRE signal. All data are presented as % of {Delta}{Psi}m observed after FCCP application. The numbers in parentheses indicate the number of cells tested.

 
In summary, the experiments shown in Fig. 4 provide evidence that the observed changes in TMRE fluorescence were due to a specific action of NO on {Delta}{Psi}m and indicate the involvement of PTP in this regulation.

Effect of mtNOS Inhibition on {Delta}{Psi}m

Mitochondrial Ca2+ uptake by respiring mitochondria induced a large membrane depolarization amounting to 82 ± 1% of the maximum decrease in TMRE fluorescence evoked by FCCP (Fig. 5A; n = 13). Cell treatment with the NOS inhibitor L-NIO (10 µM) for 4 min after cell permeabilization resulted in a partial prevention of the Ca2+-induced depolarization (Fig. 5B). In the presence of L-NIO, the magnitude of Ca2+-induced depolarization was only 66 ± 2% (P < 0.001; n = 15; Fig. 5C) compared with Ca2+-dependent depolarization in the absence of L-NIO. In addition, the initial level of fluorescence was higher in L-NIO-treated cells compared with control. In a total of 17 cells, the initial TMRE fluorescence level was 129 ± 6% of control (P < 0.01). These data suggest that mitochondrial NO production can modulate the {Delta}{Psi}m and therefore can increase or decrease the driving force for mitochondrial Ca2+ uptake.



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Fig. 5. Effect of mtNOS inhibition on {Delta}{Psi}m. A: typical trace of TMRE fluorescence after addition of 2 µM extramitochondrial Ca2+, followed by subsequent application of 1 µM FCCP. B: cells were pretreated with 10 µM of the eNOS blocker L-NIO for 4 min after cell membrane permeabilization. Shown is a typical trace recorded during Ca2+ application and withdrawal in the presence of 10 µM L-NIO. C: summary of normalized TMRE fluorescence changes produced by Ca2+ application in the absence (cntrl) and presence of L-NIO. *P < 0.001, statistically significant difference compared with control.

 
Effect of NO on Mitochondrial Permeability Transition Pore

The data in Fig. 4E suggest that NO can modulate the activity of the PTP in mitochondria of CPAE cells. This suggests that the PTP can serve as an additional mechanism for Ca2+ release during mitochondrial Ca2+ overload. To explore the effect of NO on the PTP directly, we used a method based on the observation that relatively large mitochondria-trapped molecules (such as the fluorescent probe calcein with a molecular weight of ~620 Da) can be released from isolated mitochondria (34) and from mitochondria in intact or permeabilized cells (42) after opening of the PTP. The release of calcein is associated with a decrease in fluorescence in permeabilized cells and can be blocked with cyclosporin A (42), directly suggesting that the pathway of calcein release involves the PTP. We loaded intact CPAE cells with the ester form of calcein under conditions favoring mitochondrial compartmentalization of the dye. Subsequently, cells were washed in dye-free Tyrode solution and permeabilized with digitonin (see METHODS for details). Figure 6 presents calcein fluorescence changes from permeabilized CPAE cells during application of different Sper/NO concentrations. At the end of each recording, we applied 40 µg/ml of the pore-forming antibiotic alamethicin (39) to provide a positive control for calcein release from mitochondria when the permeability of the mitochondrial membrane was increased. Loss of mitochondrial calcein was quantified as the rate of decline of fluorescence calculated from the linear fit to the decrease of fluorescence during Sper/NO application compared with the initial level of decline in fluorescence (100%). Figure 6A shows that application of 50 µM of Sper/NO did not change calcein fluorescence significantly (rate of fluorescence decline = 116 ± 9% of control; n = 9), while subsequent application of 300 µM Sper/NO induced a loss of calcein from mitochondria (229 ± 13% of control; n = 18, P < 0.001). The decrease in calcein fluorescence induced by 300 µM Sper/NO was partially prevented by cell treatment with 100 µM DQ, the PTP inhibitor (136 ± 16%; n = 5). This effect was significantly less (P < 0.01) than the effect of 300 µM Sper/NO alone. The experiment with DQ confirmed that the observed changes in calcein fluorescence during Sper/NO application were due to the opening of the PTP. Lower concentrations of Sper/NO (100 and 200 µM) also induced a decrease in calcein fluorescence, although the rate of decrease was slower (100 µM Sper/NO: 135 ± 14% of control, n = 6, P < 0.05; 200 µM Sper/NO: 207 ± 37% of control, n = 4, P < 0.01; see Fig. 6C). Figure 6D summarizes the effects of different Sper/NO concentrations on the PTP. Altogether, these data suggest that NO can trigger PTP opening, thereby providing an additional mechanism for Ca2+ release.



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Fig. 6. Effect of NO on the activity of the mitochondrial permeability transition pore. Representative traces of mitochondria-trapped calcein fluorescence changes from CPAE cells after permeabilization with 10 µM digitonin. A: application of 300 µM Sper/NO induced a decrease in calcein fluorescence while 50 µM Sper/NO had no effect. The subsequent application of the pore-forming antibiotic alamethicin (40 µg/ml) resulted in a rapid loss of calcein fluorescence, providing a positive control for maximal calcein release from the mitochondrial matrix. B: treatment with 100 µM of the PTP inhibitor decylubiquinone (DQ), significantly decreased the Sper/NO-induced loss of calcein from mitochondria. C: representative trace of calcein fluorescence changes after subsequent application of 100 and 200 µM Sper/NO. D: summary of the effects of different Sper/NO concentrations on the rate of calcein release from mitochondria. The data were normalized in each individual cell for the initial rate of fluorescence decline (100%). The numbers in parentheses indicate the number of cells tested. +P < 0.05, #P < 0.01, *P < 0.001, statistical significance. The effects of Sper/NO alone were compared with control. The effect of 300 µM Sper/NO+DQ was compared with 300 µM Sper/NO alone.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondria are essential for cellular energy metabolism and Ca2+ homeostasis. NO is known to have an inhibitory effect on cell respiration of different cell types, including vascular endothelial cells (6, 7, 18). It has been shown that endogenous NO modulates cellular O2 consumption. NO acts at the level of cytochrome oxidase, reducing the affinity of the enzyme for O2 (12, 16). Disruption of mitochondrial respiration by NO has been proposed to be partially responsible for the energy depletion in neurons (10) that led to the disruption of Ca2+ homeostasis (11). We have demonstrated previously that in vascular endothelial cells, NO inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake; however, the effect of NO on mitochondrial Ca2+ homeostasis has not been evaluated in details (19). Surprisingly, although numerous data exist on the effect of NO on cell respiration, relatively little is known about the effect of NO on mitochondrial Ca2+ homeostasis.

Mitochondria are capable of Ca2+ accumulation resulting from agonist-induced cytoplasmic [Ca2+] elevations. Ca2+ enters mitochondria via an electrogenic Ca2+ uniporter driven by the electrical potential difference across the inner mitochondrial membrane and is extruded by either Na+/Ca2+ or H+/Ca2+ exchange (41). Using fluo-3 trapped inside mitochondria, we evaluated the effect of NO on mitochondrial Ca2+ uptake through the electrogenic mitochondrial Ca2+ uniporter in digitonin-permeabilized CPAE cells. The method of cell membrane permeabilization has proved to be extremely valuable to measure the activities of mitochondria and other cellular organelles in situ without the need for isolation procedures that can adversely affect mitochondrial functions. The method has the unique advantage that it allows control of the environment surrounding mitochondria (i.e., the cytoplasm environment) and to monitor the interactions between different organelles in their native structural arrangement (22). In the present study, we demonstrated that the application of high concentrations of NO (supplied by 100–500 µM Sper/NO) resulted in mitochondrial membrane depolarization and subsequent decrease of mitochondrial Ca2+ uptake. Mitochondrial Ca2+ extrusion was also decreased in these experiments; however, we believe that this was a consequence of the inhibitory effect of Sper/NO on Ca2+ uptake and decreased [Ca2+]mt levels required for stimulation of Ca2+ extrusion mechanisms such as Na+/Ca2+ or H+/Ca2+ exchangers. Because Na+/Ca2+ or H+/Ca2+ exchangers are Ca2+ dependent, decreasing of [Ca2+]mt will result in a lowering of the rate of Ca2+ extrusion.

Low concentrations of Sper/NO (<100 µM), which generate physiological [NO] levels, had no effect on [Ca2+]mt. These results are in agreement with our previous observations in intact CPAE cells (19) that low concentrations of NO had no effect on mitochondrial Ca2+ content releasable by ionomycin. Our new data with L-arginine and L-NIO from permeabilized cells favor the possibility that mitochondrially produced NO can help regulate mitochondrial Ca2+ fluxes. In intact cells caveolae-located NOS is the main source of NO, thus [Ca2+]mt is unlikely to be controlled solely by mtNOS. Under pathological conditions, however, caveolar NOS can be compromised (see, e.g., Ref. 35) and mtNOS may serve as a compensatory mechanism to control mitochondrial Ca2+ transport. Altogether, our data indicate that the modulation of mitochondrial Ca2+ uptake by NO might play an important role in the prevention of mitochondrial Ca2+ overload. For example, it was shown that NO provided protection during simulated ischemia and reoxygenation in isolated guinea pig (31) and neonatal rat ventricular cardiomyocytes (44). The cardioprotection was mediated by the ability of NO to decrease Ca2+ sequestration into the mitochondria.

It is well established that an essential link between mitochondrial electron transport and ATP synthesis is the maintenance of {Delta}{Psi}m with negative intramitochondrial polarity. In isolated mitochondria, values of {Delta}{Psi}m on the order of –180 mV or more have been obtained during state 4 respiration (for review, see Ref. 28). In our experiments, we have investigated the effect of different NO concentrations on {Delta}{Psi}m. We found that application of Sper/NO induced a dose-dependent and reversible depolarization of the membrane potential. Moreover, inhibition of mtNOS with L-NIO led to an increased initial level of TMRE fluorescence (i.e., hyperpolarization) and partially prevented Ca2+-induced depolarizations (Fig. 5). Altogether, these data suggest that NO generated locally by mtNOS exerts a substantial control over {Delta}{Psi}m. Mitochondrial depolarization by high NO concentrations were demonstrated earlier in cultured hippocampal neurons (10) and mouse heart (5). The question was raised whether these depolarizations were mediated by NO itself or its byproduct ONOO. ONOO can inhibit enzymes of oxidative phosphorylation in isolated mitochondria or submitochondrial particles (14, 35). Membrane depolarization by NO donors was primarily mediated by NO rather than by ONOO because cell treatment with hemoglobin completely prevented the effect of NO donors and SOD did not affect the depolarization induced by NO (10). We also did not find any significant effect of the SOD mimetic and ONOO scavenger Mn-TBAP on Sper/NO-induced changes in {Delta}{Psi}m (Fig. 4, D and F). Although the effect was small and not statistically significant, we found a slightly more pronounced depolarization by 300 µM Sper/NO in the presence of Mn-TBAP, consistent with an enhanced NO production in the presence of SOD and reduced NO conversion to ONOO (20). Cell treatment with the NO scavenger PTIO, however, resulted in complete prevention of Sper/NO-induced membrane depolarization (Fig. 4, C and F). Furthermore, the effects of ONOO have been reported to be irreversible compared with those of NO, further indicating that the observed changes in {Delta}{Psi}m were due to rapid and reversible effects of NO on the respiratory chain. The effect was mediated by NO interaction with cytochrome oxidase because mitochondria treatment with sodium cyanide (blocks respiratory chain at the level of cytochrome oxidase) prevented the NO-induced depolarization of mitochondrial membrane, whereas other inhibitors of respiratory chain did not affect it (data not shown).

An alternative explanation for the decrease of [Ca2+]mt by NO is the opening of the mitochondrial permeability transition pore. In isolated mitochondria, conditions have been described that cause a Ca2+-dependent increase in mitochondrial permeability to ions and solutes with molecular weights up to 1,500 Da, matrix swelling and uncoupling of oxidative phosphorylation. The pore is protected from opening by low pH, a high electrochemical proton gradient ({Delta}{Psi}m), ADP, and pore inhibitors (such as CsA and DQ), whereas pore opening is enhanced by depleting ADP, by Pi, or by low {Delta}{Psi}m (29). The reported effects of NO on the mitochondrial PTP appear to be rather inconsistent. Both triggering and inhibitory effects of NO on PTP have been described (2, 8). NO, derived from NONOate NO donors, has also been reported to have a dual effect on PTP, depending on NO concentration. NO reversibly inhibited PTP opening with IC50 of 11 nM NO/s, whereas at supraphysiological release rates (>2 µM/s), NO accelerated PTP opening (9). Similar results were found in rat liver mitochondria, where the application of lower NO donor concentrations [1 to 20 µM of GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride), 3-morpholinosydnonimine, and SNAP] had no effect or delayed PTP opening, whereas doses from 20 to 100 µM accelerated Ca2+ overload-induced PTP (43). We used a fluorescence assay (42) to evaluate the effect of NO on the PTP. PTP opening causes matrix-trapped calcein to be released from mitochondria, which results in a decrease of mitochondrial calcein fluorescence. Because application of high concentrations of Sper/NO induced a rapid and sustained drop in membrane potential, which was partially prevented by the PTP inhibitor DQ (Fig. 4, E and F), we tested the hypothesis that the PTP is involved. Indeed, we found that the concentrations of Sper/NO (100–500 µM) that were able to induce mitochondrial membrane depolarization also initiated PTP opening. The decrease in calcein fluorescence induced by Sper/NO application was partially prevented by cell treatment with 100 µM DQ, a PTP inhibitor (23), which confirmed that the observed effect was due to opening of the PTP. The correlation between a sustained drop in {Delta}{Psi}m and PTP opening was observed previously in CPAE cells (33). Moreover, it was shown that individual mitochondria displayed repetitive opening and closing of the PTP ("flickering") spontaneously (41) as well as during exposure to oxidative stress and/or Ca2+ overload (33, 54). These short openings of PTP might serve as an emergency mechanism allowing the dissipation of {Delta}{Psi}m (and thus dramatically reducing the driving force for Ca2+ uptake), leading to fast release of accumulated Ca2+ ions and the decreased generation of endogenous oxygen radicals.

In conclusion, herein we have presented evidence that the higher levels of NO, usually reached during pathological conditions, produced either by plasma membrane-associated eNOS or locally by mitochondrially located NOS, would provide protection against mitochondrial Ca2+ overload. This protection is mediated by decreasing {Delta}{Psi}m, which leads to the decreased driving force for mitochondrial Ca2+ uptake. The reversible opening of PTP might provide an additional emergency mechanism preventing mitochondrial Ca2+ overload.


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 ABSTRACT
 METHODS
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 DISCUSSION
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-62231 (to L. A. Blatter) and American Heart Association Grant AHA-0425761Z (to E. N. Dedkova).


    ACKNOWLEDGMENTS
 
We thank Dr. Jose Puglisi for expert assistance with estimation of NO concentrations released from NO donors, as well as Holly R. Gray and Anne Pezalla for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Blatter, Dept. of Physiology, Loyola Univ. Chicago, 2160 S. First Ave., Maywood, IL 60153 (e-mail: lblatte{at}lumc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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