| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Research Communications |
a Department of Biochemical Sciences `A. Rossi Fanelli' and CNR Center of Molecular Biology, University of Rome `La Sapienza', I-00185 Roma, Italy
b CNR–Institute of Biophysics, Pisa, Italy
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: NO • cell respiration • mitochondria • cytochrome oxidase • NOR1 • neuroblastoma
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In this work we ascertained that mitochondrial respiration of rhabdomyosarcoma (RDM) and neuroblastoma (NRB) cells is controlled by NO, even when cells are grown in culture under physiological metabolic conditions. The experimental approach chosen is based on the original method by Johnson et al. (23), who introduced a way of probing the mitochondrial activity state by measuring the extent of the membrane potential-driven import of the fluorescent cationic dye rhodamine 123 (RD123). Using single cell microscopy, we found that the import of RD123 into mitochondria of cultured cells is modulated by the endogenous NO flux produced by NOS. We compared the results of bulk measurements, commonly used to detect cell fluorescence (cytofluorimetry), with single cell microscopic data and found the former to be misleading in describing changes of mitochondrial function.
Our results provide a tool for investigating NO physiopathology in single cells and to rationalize via reversible inhibition of COX the puzzling high KM for O2 observed in vivo (24, 25).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fluorescence microscopy measurements
Cells grown on coverslips were incubated for 5 min at 37°C in the culture medium containing 1 µg/ml RD123 (Sigma), washed three times with phosphate-buffered saline (PBS) and mounted on a microscope slide for fluorescence observation. When necessary, cells were incubated (typically for 5 min) with ionophores before exposure to RD123; the ionophore was maintained throughout the ensuing RD123 uptake phase. For observation, cells on coverslips were mounted on a silicon covered microscope slide in which a living chamber was obtained by removing a circular portion (1.5 cm approximately) of the silicon rubber. After washing with PBS, cells were observed under the microscope by using the filter set for fluorescein. Fluorescence signals of cells loaded with RD123 were recorded using an epifluorescence-equipped Zeiss Axiophot microscope connected to an integrating Hamamatsu C5405–01 solid-state CCD camera whose output is internally digitized in a Hamamatsu Argus 20 controller and transferred to an Intel 80486-based personal computer. Images are digitized as 256 gray levels bitmaps using standard and original software (26). Control of the integration of the fluorescence signals allowed us to quantitatively compare images obtained from different samples.
Fluorescence bulk measurements were carried out using a FACScan flow cytometer (Becton-Dickinson, San Jose, Calif.) equipped with an air-cooled 488 nm argon-ion laser. Logarithmic RD123 fluorescence was analyzed using the FL1 channel without compensation since RD123 has a single wavelength emission (27); 10,000 events were acquired for each sample, and the list mode data were analyzed using the Cellquest Becton-Dickinson software.
Stock solutions of valinomycin and nigericin (Sigma) were prepared in 99% ethanol at 2 mM concentration. +/-(E)-Metil-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR1, from Calbiochem-Novabiochem Corporation, San Diego, Calif.), dissolved in DMSO (stock solution 43 mM), was added to the cell culture medium to the final concentration of 1 mM. All other reagents were of analytical grade.
Analysis of data
Microscopic images with constant cell density (approximately six to eight cells /frame with the 40x lens and a further enlargement of approximately 20-fold on the T-mounted camera) were digitized at a constant exposure time and CCD gain (adjusted to yield a signal for the cell cytoplasm of 40 to 60 gray levels, on a full scale of 0–255). Image analysis was carried out on the frame histograms, avoiding manipulation of the images (such as contrast or profile enhancement). The histograms were calculated using a program developed with the Borland Pascal compiler and fed to the calculus package MATLAB (The Math Works Inc., South Natick, Mass.) as two columns of ASCII matrices: the first column (X) contained the full scale of gray levels (0 to 255), and the second (Y) the sum of pixels with intensity Xi. In a typical histogram, it is easy to recognize 1) a very sharp peak with minimal intensity (gray level 15 to 20), corresponding to the frame background (i.e., the area between cells); and 2) a broader peak (partially superimposed to the background) corresponding to the cell cytoplasm, which gradually merges with the high intensity, broadly distributed fluorescence assigned to mitochondria. In most frames, the threshold that excludes background (t1) and cell cytoplasm (t2) can be selected by visual inspection. Analysis of the histograms allowed us to preliminarily discard unsatisfactory (because of high background) images. After histogram deconvolution into areas corresponding to background, cell cytoplasm, and mitochondria, the following parameters were calculated: 1) the sum of pixels with intensity higher than the background (t1), which defines the area of cell cytoplasm (including mitochondria; CC), and 2) the sum of the pixels with intensity higher than the cell cytoplasm (t2), each pixel multiplied by its intensity to obtain the integrated signal of mitochondria (M). The analytical expressions are:
 |
 |
The ratio M/CC defines the normalized mitochondrial fluorescence (MF), proportional to the amount of dye imported into the mitochondria of each cell. The absolute value of MF may show fluctuations (within 20–30%) among experiments carried out in different days, due to slight changes in the microscope lamp emission, camera setup, and cell growth pattern. For this reason, a complete set of experiments was always carried out on the same day and with the identical instrumental setup. On average, eight images were collected for each experimental condition. The average values of MF and its standard deviation (SD) were calculated between frames; thus, no attempt was made to calculate the SD within frames (i.e., per cell) because this requires direct manipulation of images, which may introduce operator-dependent errors.
The mitochondrial fluorescence detected under the different conditions has been quantitatively converted into fluorescence difference (
F) with respect to the valinomycin-nigericin (background) level. Valinomycin is a K+-specific uniporter that collapses the electrical component of the mitochondrial transmembrane gradient (28); when added to the cells, rhodamine import into mitochondria is virtually zero. Nigericin is an electroneutral H+-K+ antiporter (28) that collapses the transmembrane pH and correspondingly increases 
; when added to the cells, rhodamine import is maximal (Fmax = MFnig - MFval,nig). Thus, the differential fluorescence Fx = MFx - MFval,nig allows us to calculate the fractional decrease or increase of the mitochondrial potential with respect to Fmax for each condition: RFx = Fx/Fmax. RF is the parameter most often used in this work (see 
Figs. 3, 4, and 5).
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fig. 1).
The potential-driven import of RD123 depends on the type and concentration of ionophore used to modulate the 
H+ and its components, pH and ; fluorescence is maximal in the presence of nigericin and almost nil in the presence of valinomycin and nigericin.
|
To determine the valinomycin concentration necessary to fully collapse the electrical component () of the membrane potential, RDM cells were incubated in the presence of increasing concentrations of valinomycin, up to 2 µM. As shown in Fig. 2,
the RD123 fluorescence decreases by increasing the concentration of valinomycin and becomes almost undetectable above 0.1 µM. As assessed by trypan blue staining, cells maintained their morphology and viability over the incubation period (15–30 min), even at the highest concentrations of valinomycin (of up to 2 µM, the value used throughout the experiments described here).
|
We conducted similar experiments to assess the lowest nigericin concentration at which the transmembrane proton gradient (pH) is fully converted into membrane potential (). Mitochondrial fluorescence increases with nigericin concentration only above ~0.5 µM and becomes maximal at ~5–10 µM (
Fig. 2). Whereas at lower concentrations of nigericin cells maintained the original morphology, the stability of the cells decreased with time above 5 µM. Cell damage was verified by partial detachment from the dishes, changes in morphology, and appearance of cytoplasmic granulations; eventually, positivity to trypan blue staining was also observed. On the basis of these observations, we have maintained the nigericin concentration constant at 2 µM throughout the course of the following experiments.
Time course of inhibition by exogenous NO and reversal of the effect
Inhibition of mitochondrial respiration by exogenous NO was demonstrated using the NO releaser NOR1. We independently confirmed that using deoxy Hb to detect NO, NOR1 in water releases stoichiometric amounts of NO with a half-time of 1.7 min (as indicated by Calbiochem; data not shown). Changes in mitochondrial differential fluorescence were monitored as a function of the time of incubation with NOR1. RDM cells incubated in air-equilibrated medium (270 µM O2) containing 1 mM NOR1 rapidly lose the ability to import RD123 ( Fig. 3);
the mitochondrial fluorescence is almost undetectable after 2 min incubation. If incubation with NOR1 in air is prolonged and the solution is not renovated, physiological mitochondrial activity is recovered within approximately 10 min. On the other hand, if after 5 min incubation a new aliquot of NOR1 is added, COX remains inhibited and mitochondrial fluorescence is essentially undetectable ( Fig. 3).
The recovery of mitochondrial respiration after treatment with NOR1 is fully compatible with the knowledge that in the presence of O2, inhibition of COX by NO is reversible as shown with cells, mitochondria, and the purified enzyme (see opening paragraphs). The fairly quick recovery (t1/2 
1–2 min) can be correlated with the rather fast NO dissociation rate constant from reduced COX (koff = 0.13 s-1) (22) and with NO loss from the aerated solution. From Fig. 3, the estimated first-order rate constant for recovery is k' 
0.01 s-1. This value is very similar to that calculated by others who found values ranging from ~0.01 s-1 (9) to ~0.02 s-1 (29), using different biological systems (rat liver and brain mitochondria or INS-1 pancreatic cells, respectively) and different ways of supplying NO. However, RDM cells that have recovered activity (as after ~10 min in Fig. 3) are already committed for apoptosis, which agrees with other findings (30). This was shown by assaying for apoptosis markers (oligonucleosomal DNA cleavage) (31) RDM cells subjected to a single treatment with NOR1 (as in Fig. 3) and further incubated 12 h under standard conditions (results not shown).
Inhibition of RD123 mitochondrial fluorescence by endogenous NO
This experiment is based on the idea that NOS can be specifically inhibited by L-nitroso-arginine (LNA) and/or 7-nitroindazole (7N), with subsequent decrease of steady-state NO fluxes in the cell (32). Stationary growing control RDM cells are characterized by a relative mitochondrial fluorescence RF = 0.4 (see Fig. 4);
after incubation with LNA, the value of RF rises to 0.8; if incubation is also conducted in the presence of nigericin, there is an additional increase to RF = 1.9.
The results of a parallel cytofluorimetric measurement are shown in the inset of Fig. 4. It is noteworthy that these determinations were carried out either under conditions identical to those for fluorescence microscopy or at higher concentrations of RD123 (10-fold) and/or longer incubation time (up to 20 min) in order to reproduce the conditions reported by others (27). It may be seen that, regardless of the presence of ionophores or NOS inhibitors, all fluorescence intensity profiles are almost superimposable. This shows that the effect clearly detected by single cell microscopy would have gone undetected with bulk measurements.
Similar experiments were conducted using NRB cells before and after induction of cell differentiation by retinoic acid (33); the results are reported in Fig. 5.
Before treatment with retinoic acid, the relative mitochondrial differential fluorescence of NRB cells is RF = 0.2; this increases to 1.4 by treatment with 7N in the presence of nigericin. After 24 h treatment with 1 µM retinoic acid, a clear homogeneous cell differentiation was observed (not shown); in differentiated cells, RF is 0.1 in the control and rises to 1.3 in the presence of 7N and nigericin. When both the retinoic acid-treated and untreated NRB cells were incubated for 5 min with 0.6 mM N-methyl-D-aspartate (NMDA), a known activator of NOS (5, 34), the mitochondrial fluorescence decreased to RF 0.2 (in the presence of nigericin, see Fig. 5).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The import of RD123 probes the mitochondrial membrane potential (). It may be worth recalling that, according to the general bioenergetic equation H+ = - 60 pH (28), in the presence of both valinomycin and nigericin the transmembrane potential H+ is fully collapsed and thus mitochondrial fluorescence is undetectable. The diffuse cytoplasmic fluorescence most often observed under these conditions was taken as being due to aspecific cytoplasmic uptake of RD123 ( Fig. 1D). Moreover, assuming the constancy of H+ at steady state and because of the complementarity of and pH, addition of nigericin converts the H+ gradient into electrical potential , thus yielding maximal RD123 import.
Does endogenous NO control the mitochondrial membrane potential of (cultured) cells?
In the presence of NOR1, which leads to production of NO, mitochondrial respiration as detected by mitochondrial fluorescence quickly decreases with time ( Fig. 3), consistent with the rate of hydrolysis of NOR1 and, thus, with the increase of NO concentration in the cell-suspending medium. In air, the NO released disappears from solution and mitochondrial respiration is restored, as shown by the import of RD123, which is fully recovered 10 min after the addition of NOR1. The time course of recovery with k' 0.01 s-1 is slower than the intrinsic NO dissociation measured independently by using purified COX (22), but similar to other determinations (9, 29).
The interconversion of pH into induced by 2 µM nigericin always leads to a substantial increase of F over the control (2.5-fold in RDM cells and 5-fold in NRB cells; Figs. 4 and 5). More interesting is that inhibition of NOS by 7N and LNA or stimulation by NMDA lead to substantial increase or decrease of mitochondrial fluorescence, respectively ( Figs. 4 and 5). Cell differentiation is known to be coupled to morphological and functional changes of mitochondria (35); therefore, we have extended our investigation to NRB cells subjected to treatment with retinoic acid, used to induce differentiation.
On the basis of previous findings (15–17) and the recent demonstration of a mitochondrial localization of NO synthase (36), depression of mitochondrial respiration by stimulation of NOS with NMDA was expected. The new observation reported here is that treatment of stationary growing cells with inhibitors of NOS leads to an increase in mitochondrial fluorescence (of about 30–50% with respect to the conditions where nigericin alone is present, as in Figs. 4 and 5). This result strongly suggests that stationary growing cultured cells are characterized by a basal NOS activity that produces NO at a concentration suitable to control electron flux through the respiratory chain.
Results were comparable in RDM and NRB cells, in the latter case, even after differentiation by retinoic acid–thus suggesting that the steady-state production of NO is independent of the cell line and differentiation degree.
Single cell microscopy vs. bulk cytofluorimetry
Even though fluorescence quantitation is particularly complex when dealing with spot-like mitochondrial emission, the single cell microscopic approach proves to have unique advantages over the widely used bulk cytofluorimetry: 1) it allows direct evaluation of the distribution of fluorescence in subcellular compartments, clearly distinguishing diffused cytoplasmic from concentrated mitochondrial signals, both of which give similar cytofluorimetric results at equal fluorochrome concentration, and 2) it is applicable to microscopic specimens of tissue, such as a biopsy. To the contrary, bulk cytofluorimetry is unsuitable for correct assignment of fluorescence to a given cell compartment, which explains why a behavior coherent with mitochondrial energization state failed to be detected by cytofluorimetry (27). In fact, we observed that all cytofluorimetric patterns determined in parallel with microscopic measurements were similar to each other (inset to Fig. 4).
Physiological relevance of O2 and NO fluxes to cytochrome oxidase activity
Brown (19) suggests that a stationary level of NO inhibiting a finite fraction of COX may provide a reasonable explanation for the ~10- to 20- fold higher KM for O2 measured for cells and tissues (KM=5–10 µM), as compared with the purified enzyme (KM=0.5 µM) (24, 25). The results reported in this paper show that cultured cells, without external NOS stimulation, steadily produce NO at a concentration suitable to depress respiration by inhibiting COX.
What is the NO concentration required to account for our observations? Evidence available on the kinetics of O2 and NO binding to COX (21, 22) may be used to attempt a calculation of consistency for basal NO flux. Assuming a simplified Michaelis scheme in which NO competes with O2 for binding to the reduced heme a3-CuB binuclear site (22, 37), it may be calculated that ~60 nM NO would raise the KM for O2 to 10 µM, the value reported for cells (24).
Finally, we notice that, in agreement with Hortelano et al. (30), cells treated with exogenous NO underwent activation of apoptosis. One may believe that endogenous NO (produced by basal NOS activity) and exogenous NO (released by NOR1) act through one and the same mechanism. In both cases, the decrease in membrane potential seen via mitochondrial import of RD123 is caused by inhibition of the respiratory chain by NO; however, in the latter case apoptosis is triggered (30). Therefore, we are faced with a puzzle: is complete inhibition of COX (as in the presence of NOR1) necessary to trigger apoptosis or is apoptosis a side effect of exposure of cellular components other than the respiratory chain to concentrations of NO higher than those metabolically produced to control respiration. These two possibilities may be amenable to experimental verification and will be further explored.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Abbreviations: COX, cytochrome c oxidase; FCS, fetal calf serum; NOS, nitric oxide synthase; RDM, rhabdomyosarcoma; NRB, neuroblastoma; RD123, rhodamine 123; PBS, phosphate-buffered saline; NOR1, +/- (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide; 7N, 7-nitroindazole; LNA, L-nitroso-arginine; NMDA, N-methyl-D-aspartate.
Received for publication April 3, 1998.
Revision received September 14, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77–82[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. T. Gladwin and D. B. Kim-Shapiro The functional nitrite reductase activity of the heme-globins Blood, October 1, 2008; 112(7): 2636 - 2647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Cooper and C. Giulivi Nitric oxide regulation of mitochondrial oxygen consumption II: molecular mechanism and tissue physiology Am J Physiol Cell Physiol, June 1, 2007; 292(6): C1993 - C2003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kakoki, R. W. McGarrah, H.-S. Kim, and O. Smithies Bradykinin B1 and B2 receptors both have protective roles in renal ischemia/reperfusion injury PNAS, May 1, 2007; 104(18): 7576 - 7581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kann and R. Kovacs Mitochondria and neuronal activity Am J Physiol Cell Physiol, February 1, 2007; 292(2): C641 - C657. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Palacios-Callender, M. Quintero, V. S. Hollis, R. J. Springett, and S. Moncada Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase PNAS, May 18, 2004; 101(20): 7630 - 7635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. F. W. HORN, G. WOLF, S. DUFFY, S. WEISS, G. KEILHOFF, and B. A. MacVICAR Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons FASEB J, October 1, 2002; 16(12): 1611 - 1622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Shimizu, Z. Lacza, N. Rajapakse, T. Horiguchi, J. Snipes, and D. W. Busija MitoKATP opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1005 - H1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Kanai, L. L. Pearce, P. R. Clemens, L. A. Birder, M. M. VanBibber, S.-Y. Choi, W. C. de Groat, and J. Peterson Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection PNAS, November 20, 2001; 98(24): 14126 - 14131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. HORTELANO, A. M. ALVAREZ, and L. BOSCA Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages FASEB J, December 1, 1999; 13(15): 2311 - 2317. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 
) or renovated (
) after 5 min (arrow), and further incubated. X axis values of data points indicate the incubation time with NOR1 before cell washing and addition of RD123. As in Fig. 2, data represent the average of at least eight independent measurements (SD 
