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Increased uptake and retention of rhodamine 123 by mitochondria of old human fibroblasts
Mechanisms of Ageing and Development, 39 (1987) 1-9 Elsevier Scientific Publishers Ireland Ltd.
1
INCREASED UPTAKE AND RETENTION OF RHODAMINE 123 BY MITOCHONDRIA OF OLD HUMAN FIBROBLASTS
ANDREW O. MARTINEZ*, CORDELIA VARA and JACQUELINE CASTRO Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285(U.S.A.) (Received January 21st, 1986) (Revision received February 5th, 1987)
SUMMARY
Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R I23-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.
Key words: Ageing; Mitochondria; Rhodamine 123; Human fibroblast
INTRODUCTION
Mitochondria are semi-autonomous, energy-generating organelles in eucaryotic cells. They are also sites for continuous damage-producing free radical reactions during respiration [ 1-3]. Hence, mitochondria could be important structures in maintaining intracellular homeostasis. Further, several investigators have proposed that
*To whom correspondence should be addressed. 0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 Elsevier Scientific Publishers Ireland Ltd.
mitochondrial degeneration may be the basic cause of cellular aging [ 1,2]. Despite a wealth of information, however, the role played by mitochondria in the aging process of mammalian cells remains controversial and uncertain. Recently [4], we reported that old human diploid fibroblasts (HF) bind more mitochondria-specific dye Rhodamine 123 (R123) than young HF. The mechanism for this increased binding of R123 is not known but could be dependent on the magnitude of the transmembrane potential maintained in the mitochondria [5 9]. The present study was conducted to evaluate whether this difference in dye uptake between young and old HF is indeed at the level of the mitochondria. Young and old HF were exposed to drugs known to perturb either transmembrane potential or metabolism of mitochondria [6,7]. These drugs produced different effects on the R123-mitochondria interaction of young and old HF indicating a difference in the magnitude of the mitochondrial transmembrane potential. Moreover, this difference could reflect an age-related change in the structure and/or function of the mitochondria. METHODS AND MATERIALS Cells and cell culture conditions The human diploid fibroblast line (75-69) used in these studies was obtained from the laboratory of Dr. George M. Martin, Department of Pathology, University of Washington, from a foreskin explant from a phenotypically normally Caucasian infant. Stock cultures were maintained in 150-cm z plastic tissue culture flasks with Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with glutamine (0.1 M), sodium pyruvate (1 mM), nonessential amino acids (100x, Gibco), penicillin ( 100 U/ml), streptomycin ( 100 ~g/ml) and 10% heat-inactivated fetal bovine serum, and buffered with HEPES (25 mM) and sodium bicarbonate (9 mM). All cultures were maintained in an atmosphere of approximately I% CO 2 in air at 37 C. Assays for mycoplasma by aerobic and anerobic cultures [10] were negative. Chemicals Stock solutions of R123 (Eastman) were prepared in double-distilled water at a concentration of 1 mg/ml. A final concentration of 0.5 #g/ml in DMEM was prepared just prior to exposure to experimental cultures. Drugs that had previously been shown to affect the transmembrane potential or metabolism of mitochondria were selected [6,7]. Valinomycin and rotenone (Sigma) were prepared as stock solutions of 1/~g/mi and 1 mM, respectively, in 95% ethanol. Final concentrations of 5 #g/ml and 10 5 M respectively, in DMEM were prepared prior to exposure to experimental cultures. Nigericin (provided by Dr. W.E. Scott of Hoffman-LaRoche, Nutley, N J) was prepared as a stock solution of 1 mg/ml in 95% ethanol. A final concentration of 5/~g/ml in DMEM was prepared just prior to exposure to experimental cultures.
Flow cytornetry Cells were harvested from subconfluent stock cultures and seeded into 75-cm 2 tissue culture flasks at a concentration of 5 × 105 cells/flask in 10 ml DMEM. After a 12-h incubation period at 37r~C, the medium was replaced with medium containing R123 (0.5/ag/ml) and the cultures incubated for an additional 30 min at 3TIC. Mitochondrial fluorescence was monitored immediately following the exposure period and at 30 and 60 min and 2 and 5 h after exposure to determine the fate of the dye. The drugs nigericin, valinomycin and rotenone were used to characterize the R123-mitochondria interaction in young and old HF. The effects of such treatments on mitochondrial uptake of R 123 were assessed after a combined R123-drug exposure period of 30 min. Drug effects on mitochondrial retention of the dye on prestained cells were assessed after.drug exposure periods of 30 and 60 min and 2 and 5 h. Fluorescence was measured by flow cytometry analysis using a FACS 420 fluorescence-activated cell sorter (Becton-Dickinson, Mountain View, CA). The 514.5-nm line from an argon laser was used. Electronic amplifier and gain settings were set such that a majority of cells in the control samples were placed in channels 128-145 (i.e. fluorescence channels i-18). Data on low angle light scatter (channels 1-127) and fluorescence intensity (channels 128-255) were displayed as separate histograms, and dead cells were "gated" out on the basis of their characteristic low light scatter profile. Data in correlated list-mode fashion were transferred to a DEC 11/23 computer (Digital Equipment Co.) for storage and statistical analysis. A plot of relative fluorescence vs. cell number was then generated for each sample. Data analysis Mean relative fluorescence (average of the distribution) was determined at identical amplifier and gain settings for both young and old HF. Thus, the relative effects of the various drugs on the Ri23-mitochondria interaction of the two cell types can be compared. Strictly, what is compared is the per cent change in mean fluorescence channel of experimental populations relative stained controls. These standardized results were then presented as plots of relative fluorescence vs. incubation time for each drug. RESULTS Effects o f drugs on R 123 uptake The 75-69 fibroblast line exhibits a n / n vitro lifespan of approximately 52 cumulative population doublings. Cells of two different population doubling levels (PDL), PDL-6 ( " y o u n g " ) and PDL-41 ("old"), were used. To assess whether the difference in R123 fluorescence between young and old H F reflected a difference in magnitude of mitochondriai transmembrane potential, the effects of drugs known to disrupt transmembrane potential or metabolism of mitochondria on the R123-mitochondria interaction of young and old H F were
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Fig. 1. FACS fluorescence distributions showing effects of drugs on uptake of R123 by young (PDL O) and old (PDL 41) HF. Cells were stained, exposed to drugs and analyzed as described in Materials and Methods. Panels a,c,e,g, represent distributions of young HF; panels b,d,f,h represent distributions of old HF. Panels a,b, uptake in drug-free medium. Panels c,d, uptake in nigericin-containing medium. Panels e,f, uptake in valinomycin-containing medium. Panels g,h, uptake in rotenone-containing medium. Mean CH. indicates the average of the fluorescence distribution.
compared. These included the ionophores nigericin and valinomycin and the inhibitor of electron transport rotenone. Distributions of fluorescence for populations stained in the presence and absence of drugs are presented in Fig. 1. As we have reported previously [4], in the absence of drugs old HF accumulated more R123 than young HF (Figs. I a,b). Further, fluorescence profiles of old HF exhibited two distinct cell subpopulations; a relatively homogeneous, low-fluorescing one, and a highly heterogeneous, brightly-fluorescing one (Fig. i b). Relative effects of the drugs on R123 uptake were assessed by comparing the shift in mean fluorescence (Mean CH.) of experimental populations to that of drug-free R123-stained controls. All three drugs modulated dye uptake; however, differences between young and old HF were evident. Nigericin, which is known to increase the transmembrane potential of mitochondria [6,7], had no apparent effect on young HF (Fig. lc) but increased Mean CH. of old HF by approximately 8% (Fig. ld). In contrast, valinomycin, which has been reported to dissipate the potential of mitochondria [6], decreased Mean CH. of young HF by approximately 24% (Fig. le), but had no apparent effect on old HF (Fig. lf). Rotenone, which inhibits electron transport required for the maintenance of the proton gradient across mitochondrial membranes [6], produced conti'asting effects; it decreased Mean CH. of young HF by over 40% (Fig. lg), but increased Mean CH. of old HF by about 10% (Fig. lh). In addition, rotenone had differential effects on the two old HF subpopulations; the low-fluorescing subpopulation increased slightly in proportion, while the brightlyfluorescing one increased in heterogeneity. Moreover, changes in fluorescence intensity (data not shown) consistent with these results also were observed by epifluorescent microscopy on living cells after combined R l23-drug treatment. Effects of drugs on R 123 retention Upon incubation of stained cells in dye-free medium, a loss of fluorescence occurred. This was due to a gradual release of the dye by the cells. Distributions of fluorescence for control populations (stained and incubated in drug-free medium) of young and old HF are presented in Fig. 2. These distributions show a more rapid loss of fluorescence by young than by old HF. Moreover, both old HF subpopulations exhibited decreases in fluorescence. Loss of fluorescence (dye release) was assessed by comparing changes in Mean CH. of incubated experimental populations relative unincubated controls. Comparisons of the kinetics of dye release (loss of fluorescence) (Fig. 3) show marked differences in the rate and pattern of release between young and old HF. As is indicated, by 30 min mean fluorescence of young HF had decreased noticeably (12%), while that of old HF had remained stable. By the end of the 5-h incubation period, mean fluorescence of young HF had decreased by approximately 50%, while that of old HF had decreased only by about 30%. To further characterize the R123-mitochondria interaction in young and old HF, the kinetics of dye release in the presence of the three drugs were also compared (Fig. 4). Relative effects of the drugs on dye retention by young and old HF were assessed by comparing the per cent change in Mean CH. of experimental populations to unincubated drug-free controls. Although all three drugs had some effect on dye release, distinct differences between young and old HF were evident. Nigericin slowed
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Fig. 2. FACS fluorescence distributions showing effects of drugs on retention of RI23 by prestained young (PDL 6) and old (PDL 41) HF. Cells were stained, incubated and analyzed as described in Materials and Methods. Panels a,c,e,g represent distributions of young HF; panels b,d,f,h represent distributions of old HF. Panels a,b, 60-rain incubation in drug-free medium. Panels c,d, 60-min incubation in nigericincontaining medium. Panels, e,f, 60-min incubation in valinomycin-containing medium. Panels g,h, 60-min incubation in rotenone-containing medium. Mean CH. indicates the average of the fluorescence distribution.
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Fig. 3. Kinetics of dye-release by young (PDL 6) and old (PDL 41) HF incubated for 30 and 60 rain and 2 and 5 h in drug-free medium. Cells were stained, incubated and analyzed as described in Materials and Methods. O O, old HF; • • , young HF.
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Fig. 4. Kinetics of dye-release by young (PDL 6) and old (PDL 41) HF incubated for 30 and 60 min and 2 and 5 h in the presence of drugs. Cells were stained, incubated and analyzed as described in Materials and Methods. Panel A, incubation in nigericin-containing medium. Panel B, incubation in valinomycincontaining medium. Panel C, incubation in rotenone-containing medium. O O, old HF; • •, young HF.
down the rate of release for both cell types but did not alter the patterns (Fig. 4A). In contrast, valinomycin markedly increased the rate of release for both cell types (Fig. 4B). Rotenone affected both the rate and pattern of release for both cell types but had a more pronounced effect on young HF. Loss of fluorescence by old HF was not evident until 60 min post-incubation, then release occurred at an accelerated rate but slowed down after 2 h. The total amount released by young HF over the 5-h time period was approximately 80% as compared to approximately 40% by old HF. Moreover, both old HF subpopulations seemed to be affected (data not shownl, however, the low-fluorescing subpopulation appeared to release the dye more slowly.
DISCUSSION Previous studies [4] have shown that old HF accumulate more mitochondriaspecific dye R123 than young HF. Although the basis for this difference remains unknown, several possible explanations can be considered: (1) a difference in mitochondrial density; (2) a difference in mitochondrial mass; (3) a difference in magnitude of mitochondrial transmembrane potential; or (4) a combination of the above. However, selective binding of R 123 by other mammalian cells has been shown to be dependent on the transmembrane potential maintained in functional mitochondria [6-8]. In this study, we have demonstrated that drugs known to disrupt the transmembrane potential of mitochondria produce different effects on young and old HF indicating a difference in magnitude of mitochondrial transmembrane potential. These results, therefore, suggest that old HF bind and retain more of the cationic dye R123 because they maintain a higher (more negative inside) mitochondrial transmembrane potential. Further, although the basis and biological significance of the two subpopulations in old HF cultures are not known, it is possible that they reflect other age-related changes in mitochondria such as an increase in mitochondrial mass. This possibility is suggested by an increase in mitochondrial branching observed in old HF. Finally, although the relation between mitochondrial transmembrane potential and cell aging remains to be determined, it is possible that a change in transmembrane potential reflects a change(s) in structure and/or function of mitochondria. Age-specific changes in membrane structure and physiological functions of mitochondria have been reported [11-13]. Further, several investigators have proposed that mitochondria are sites for continuous damage-producing free radical reactions during the course of normal respiration [ 1-3]. Therefore, it is possible that damage to mitochondrial membranes be reflected in changes in magnitude of transmembrane potential of mitochondria and could have an accumulative deleterious effect leading to a decline in mitochondrial functional capacity. Further, Miquel et al. [2] suggest that there is no reason to rule out mitochondrial degeneration as "the primary cause of all other manifestations of senescence". Clearly, further studies are needed to determine the significance of these results to mitochondrial structure and/or function, and to the phenomenon of cellular aging.
ACKNOWLEDGEMENTS W e wish to t h a n k M r s . A d e l i n e R u s h for e x p e r t s e c r e t a r i a l assistance. T h i s s t u d y was s u p p o r t e d by N a t i o n a l I n s t i t u t e s o f H e a l t h g r a n t s R R 0 8 1 9 4 a n d G M 0 7 7 1 7 a n d N a t i o n a l Science F o u n d a t i o n g r a n t 8003728. REFERENCES I D. Harmon, The aging process. Proc. Natl. Acad. Sci. U.S.A., 78(1981) 7124-7128. 2 J. Miquel, A.D. Economos, J. Fleming and J.E. Johnson, Mitochondrial role in cell aging, Exp. Gerontol., 15 (1980) 575 59t. 3 J. Miquel and J.E. Fleming, A two-step hypothesis on the mechanisms of in vitro cell aging: Cell differentiation followed by intrinsic mitochondrial mutagenesis. Exp. Gerontol., 19 (1984) 31-36. 4 A.O. Martinez, A. Vigil and J. Vila, Flow cytometric analysis of mitochondria-associated fluorescence in young and old human fibroblasts. Exp. Cell Res., 164 (1986) 551-555. 5 L.V. Johnson, M.L. Walsh and L.B. Chen, Localization ofmitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. U.S.A., 77(1980)99(~994. 6 L.V. Johnson, M.L. Walsh, B.J. Bockus and L.B. Chen, Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol., 88 (1981) 526~535. 7 L.V. Johnson, I.C. Summerhayes and L.B. Chen, Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells. Cell, 28 (1982) 7-14. 8 S. Goldstein and L.B. Korczack, Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123. J. Cell Biol., 91 (1981) 392-398. 9 H.M. Shapiro, P.J. Natale and L.A. Kamentsky, Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proe. Natl. Acad. Sci. U.S.A., 76 (1979) 5728-5730. I0 G.E. Kenny, Serological comparison of ten glycolytic mycoplasma species. J. Bacteriol., 98 (1969) 1044~1055. I I F. Schroeder, I Goetz and E. Roberts, Age-related alterations in cultured human fibroblast membrane structure and function. Mech. Ageing Dev., 25 (1984) 365-389. 12 S. Goldstein, E.J. Moerman and K. Porter, High-voltage electron microscopy of human diploid fibroblasts during aging/n vitro: Morphometric analysis of mitochondria. Exp. Cell. Res., 154 (1984) 101 III. 13 S. Goldstein, S.R. Ballantyne, A.L. Robson and E.J. Moerman, Energy metabolism in cultured human fibroblasts during aging in vitro. J. Cell. Physiol., 112 (1982) 112~419.
1
INCREASED UPTAKE AND RETENTION OF RHODAMINE 123 BY MITOCHONDRIA OF OLD HUMAN FIBROBLASTS
ANDREW O. MARTINEZ*, CORDELIA VARA and JACQUELINE CASTRO Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285(U.S.A.) (Received January 21st, 1986) (Revision received February 5th, 1987)
SUMMARY
Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R I23-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.
Key words: Ageing; Mitochondria; Rhodamine 123; Human fibroblast
INTRODUCTION
Mitochondria are semi-autonomous, energy-generating organelles in eucaryotic cells. They are also sites for continuous damage-producing free radical reactions during respiration [ 1-3]. Hence, mitochondria could be important structures in maintaining intracellular homeostasis. Further, several investigators have proposed that
*To whom correspondence should be addressed. 0047-6374/87/$03.50 Printed and Published in Ireland
© 1987 Elsevier Scientific Publishers Ireland Ltd.
mitochondrial degeneration may be the basic cause of cellular aging [ 1,2]. Despite a wealth of information, however, the role played by mitochondria in the aging process of mammalian cells remains controversial and uncertain. Recently [4], we reported that old human diploid fibroblasts (HF) bind more mitochondria-specific dye Rhodamine 123 (R123) than young HF. The mechanism for this increased binding of R123 is not known but could be dependent on the magnitude of the transmembrane potential maintained in the mitochondria [5 9]. The present study was conducted to evaluate whether this difference in dye uptake between young and old HF is indeed at the level of the mitochondria. Young and old HF were exposed to drugs known to perturb either transmembrane potential or metabolism of mitochondria [6,7]. These drugs produced different effects on the R123-mitochondria interaction of young and old HF indicating a difference in the magnitude of the mitochondrial transmembrane potential. Moreover, this difference could reflect an age-related change in the structure and/or function of the mitochondria. METHODS AND MATERIALS Cells and cell culture conditions The human diploid fibroblast line (75-69) used in these studies was obtained from the laboratory of Dr. George M. Martin, Department of Pathology, University of Washington, from a foreskin explant from a phenotypically normally Caucasian infant. Stock cultures were maintained in 150-cm z plastic tissue culture flasks with Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with glutamine (0.1 M), sodium pyruvate (1 mM), nonessential amino acids (100x, Gibco), penicillin ( 100 U/ml), streptomycin ( 100 ~g/ml) and 10% heat-inactivated fetal bovine serum, and buffered with HEPES (25 mM) and sodium bicarbonate (9 mM). All cultures were maintained in an atmosphere of approximately I% CO 2 in air at 37 C. Assays for mycoplasma by aerobic and anerobic cultures [10] were negative. Chemicals Stock solutions of R123 (Eastman) were prepared in double-distilled water at a concentration of 1 mg/ml. A final concentration of 0.5 #g/ml in DMEM was prepared just prior to exposure to experimental cultures. Drugs that had previously been shown to affect the transmembrane potential or metabolism of mitochondria were selected [6,7]. Valinomycin and rotenone (Sigma) were prepared as stock solutions of 1/~g/mi and 1 mM, respectively, in 95% ethanol. Final concentrations of 5 #g/ml and 10 5 M respectively, in DMEM were prepared prior to exposure to experimental cultures. Nigericin (provided by Dr. W.E. Scott of Hoffman-LaRoche, Nutley, N J) was prepared as a stock solution of 1 mg/ml in 95% ethanol. A final concentration of 5/~g/ml in DMEM was prepared just prior to exposure to experimental cultures.
Flow cytornetry Cells were harvested from subconfluent stock cultures and seeded into 75-cm 2 tissue culture flasks at a concentration of 5 × 105 cells/flask in 10 ml DMEM. After a 12-h incubation period at 37r~C, the medium was replaced with medium containing R123 (0.5/ag/ml) and the cultures incubated for an additional 30 min at 3TIC. Mitochondrial fluorescence was monitored immediately following the exposure period and at 30 and 60 min and 2 and 5 h after exposure to determine the fate of the dye. The drugs nigericin, valinomycin and rotenone were used to characterize the R123-mitochondria interaction in young and old HF. The effects of such treatments on mitochondrial uptake of R 123 were assessed after a combined R123-drug exposure period of 30 min. Drug effects on mitochondrial retention of the dye on prestained cells were assessed after.drug exposure periods of 30 and 60 min and 2 and 5 h. Fluorescence was measured by flow cytometry analysis using a FACS 420 fluorescence-activated cell sorter (Becton-Dickinson, Mountain View, CA). The 514.5-nm line from an argon laser was used. Electronic amplifier and gain settings were set such that a majority of cells in the control samples were placed in channels 128-145 (i.e. fluorescence channels i-18). Data on low angle light scatter (channels 1-127) and fluorescence intensity (channels 128-255) were displayed as separate histograms, and dead cells were "gated" out on the basis of their characteristic low light scatter profile. Data in correlated list-mode fashion were transferred to a DEC 11/23 computer (Digital Equipment Co.) for storage and statistical analysis. A plot of relative fluorescence vs. cell number was then generated for each sample. Data analysis Mean relative fluorescence (average of the distribution) was determined at identical amplifier and gain settings for both young and old HF. Thus, the relative effects of the various drugs on the Ri23-mitochondria interaction of the two cell types can be compared. Strictly, what is compared is the per cent change in mean fluorescence channel of experimental populations relative stained controls. These standardized results were then presented as plots of relative fluorescence vs. incubation time for each drug. RESULTS Effects o f drugs on R 123 uptake The 75-69 fibroblast line exhibits a n / n vitro lifespan of approximately 52 cumulative population doublings. Cells of two different population doubling levels (PDL), PDL-6 ( " y o u n g " ) and PDL-41 ("old"), were used. To assess whether the difference in R123 fluorescence between young and old H F reflected a difference in magnitude of mitochondriai transmembrane potential, the effects of drugs known to disrupt transmembrane potential or metabolism of mitochondria on the R123-mitochondria interaction of young and old H F were
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compared. These included the ionophores nigericin and valinomycin and the inhibitor of electron transport rotenone. Distributions of fluorescence for populations stained in the presence and absence of drugs are presented in Fig. 1. As we have reported previously [4], in the absence of drugs old HF accumulated more R123 than young HF (Figs. I a,b). Further, fluorescence profiles of old HF exhibited two distinct cell subpopulations; a relatively homogeneous, low-fluorescing one, and a highly heterogeneous, brightly-fluorescing one (Fig. i b). Relative effects of the drugs on R123 uptake were assessed by comparing the shift in mean fluorescence (Mean CH.) of experimental populations to that of drug-free R123-stained controls. All three drugs modulated dye uptake; however, differences between young and old HF were evident. Nigericin, which is known to increase the transmembrane potential of mitochondria [6,7], had no apparent effect on young HF (Fig. lc) but increased Mean CH. of old HF by approximately 8% (Fig. ld). In contrast, valinomycin, which has been reported to dissipate the potential of mitochondria [6], decreased Mean CH. of young HF by approximately 24% (Fig. le), but had no apparent effect on old HF (Fig. lf). Rotenone, which inhibits electron transport required for the maintenance of the proton gradient across mitochondrial membranes [6], produced conti'asting effects; it decreased Mean CH. of young HF by over 40% (Fig. lg), but increased Mean CH. of old HF by about 10% (Fig. lh). In addition, rotenone had differential effects on the two old HF subpopulations; the low-fluorescing subpopulation increased slightly in proportion, while the brightlyfluorescing one increased in heterogeneity. Moreover, changes in fluorescence intensity (data not shown) consistent with these results also were observed by epifluorescent microscopy on living cells after combined R l23-drug treatment. Effects of drugs on R 123 retention Upon incubation of stained cells in dye-free medium, a loss of fluorescence occurred. This was due to a gradual release of the dye by the cells. Distributions of fluorescence for control populations (stained and incubated in drug-free medium) of young and old HF are presented in Fig. 2. These distributions show a more rapid loss of fluorescence by young than by old HF. Moreover, both old HF subpopulations exhibited decreases in fluorescence. Loss of fluorescence (dye release) was assessed by comparing changes in Mean CH. of incubated experimental populations relative unincubated controls. Comparisons of the kinetics of dye release (loss of fluorescence) (Fig. 3) show marked differences in the rate and pattern of release between young and old HF. As is indicated, by 30 min mean fluorescence of young HF had decreased noticeably (12%), while that of old HF had remained stable. By the end of the 5-h incubation period, mean fluorescence of young HF had decreased by approximately 50%, while that of old HF had decreased only by about 30%. To further characterize the R123-mitochondria interaction in young and old HF, the kinetics of dye release in the presence of the three drugs were also compared (Fig. 4). Relative effects of the drugs on dye retention by young and old HF were assessed by comparing the per cent change in Mean CH. of experimental populations to unincubated drug-free controls. Although all three drugs had some effect on dye release, distinct differences between young and old HF were evident. Nigericin slowed
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Fig. 2. FACS fluorescence distributions showing effects of drugs on retention of RI23 by prestained young (PDL 6) and old (PDL 41) HF. Cells were stained, incubated and analyzed as described in Materials and Methods. Panels a,c,e,g represent distributions of young HF; panels b,d,f,h represent distributions of old HF. Panels a,b, 60-rain incubation in drug-free medium. Panels c,d, 60-min incubation in nigericincontaining medium. Panels, e,f, 60-min incubation in valinomycin-containing medium. Panels g,h, 60-min incubation in rotenone-containing medium. Mean CH. indicates the average of the fluorescence distribution.
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Fig. 3. Kinetics of dye-release by young (PDL 6) and old (PDL 41) HF incubated for 30 and 60 rain and 2 and 5 h in drug-free medium. Cells were stained, incubated and analyzed as described in Materials and Methods. O O, old HF; • • , young HF.
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Fig. 4. Kinetics of dye-release by young (PDL 6) and old (PDL 41) HF incubated for 30 and 60 min and 2 and 5 h in the presence of drugs. Cells were stained, incubated and analyzed as described in Materials and Methods. Panel A, incubation in nigericin-containing medium. Panel B, incubation in valinomycincontaining medium. Panel C, incubation in rotenone-containing medium. O O, old HF; • •, young HF.
down the rate of release for both cell types but did not alter the patterns (Fig. 4A). In contrast, valinomycin markedly increased the rate of release for both cell types (Fig. 4B). Rotenone affected both the rate and pattern of release for both cell types but had a more pronounced effect on young HF. Loss of fluorescence by old HF was not evident until 60 min post-incubation, then release occurred at an accelerated rate but slowed down after 2 h. The total amount released by young HF over the 5-h time period was approximately 80% as compared to approximately 40% by old HF. Moreover, both old HF subpopulations seemed to be affected (data not shownl, however, the low-fluorescing subpopulation appeared to release the dye more slowly.
DISCUSSION Previous studies [4] have shown that old HF accumulate more mitochondriaspecific dye R123 than young HF. Although the basis for this difference remains unknown, several possible explanations can be considered: (1) a difference in mitochondrial density; (2) a difference in mitochondrial mass; (3) a difference in magnitude of mitochondrial transmembrane potential; or (4) a combination of the above. However, selective binding of R 123 by other mammalian cells has been shown to be dependent on the transmembrane potential maintained in functional mitochondria [6-8]. In this study, we have demonstrated that drugs known to disrupt the transmembrane potential of mitochondria produce different effects on young and old HF indicating a difference in magnitude of mitochondrial transmembrane potential. These results, therefore, suggest that old HF bind and retain more of the cationic dye R123 because they maintain a higher (more negative inside) mitochondrial transmembrane potential. Further, although the basis and biological significance of the two subpopulations in old HF cultures are not known, it is possible that they reflect other age-related changes in mitochondria such as an increase in mitochondrial mass. This possibility is suggested by an increase in mitochondrial branching observed in old HF. Finally, although the relation between mitochondrial transmembrane potential and cell aging remains to be determined, it is possible that a change in transmembrane potential reflects a change(s) in structure and/or function of mitochondria. Age-specific changes in membrane structure and physiological functions of mitochondria have been reported [11-13]. Further, several investigators have proposed that mitochondria are sites for continuous damage-producing free radical reactions during the course of normal respiration [ 1-3]. Therefore, it is possible that damage to mitochondrial membranes be reflected in changes in magnitude of transmembrane potential of mitochondria and could have an accumulative deleterious effect leading to a decline in mitochondrial functional capacity. Further, Miquel et al. [2] suggest that there is no reason to rule out mitochondrial degeneration as "the primary cause of all other manifestations of senescence". Clearly, further studies are needed to determine the significance of these results to mitochondrial structure and/or function, and to the phenomenon of cellular aging.
ACKNOWLEDGEMENTS W e wish to t h a n k M r s . A d e l i n e R u s h for e x p e r t s e c r e t a r i a l assistance. T h i s s t u d y was s u p p o r t e d by N a t i o n a l I n s t i t u t e s o f H e a l t h g r a n t s R R 0 8 1 9 4 a n d G M 0 7 7 1 7 a n d N a t i o n a l Science F o u n d a t i o n g r a n t 8003728. REFERENCES I D. Harmon, The aging process. Proc. Natl. Acad. Sci. U.S.A., 78(1981) 7124-7128. 2 J. Miquel, A.D. Economos, J. Fleming and J.E. Johnson, Mitochondrial role in cell aging, Exp. Gerontol., 15 (1980) 575 59t. 3 J. Miquel and J.E. Fleming, A two-step hypothesis on the mechanisms of in vitro cell aging: Cell differentiation followed by intrinsic mitochondrial mutagenesis. Exp. Gerontol., 19 (1984) 31-36. 4 A.O. Martinez, A. Vigil and J. Vila, Flow cytometric analysis of mitochondria-associated fluorescence in young and old human fibroblasts. Exp. Cell Res., 164 (1986) 551-555. 5 L.V. Johnson, M.L. Walsh and L.B. Chen, Localization ofmitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. U.S.A., 77(1980)99(~994. 6 L.V. Johnson, M.L. Walsh, B.J. Bockus and L.B. Chen, Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol., 88 (1981) 526~535. 7 L.V. Johnson, I.C. Summerhayes and L.B. Chen, Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells. Cell, 28 (1982) 7-14. 8 S. Goldstein and L.B. Korczack, Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123. J. Cell Biol., 91 (1981) 392-398. 9 H.M. Shapiro, P.J. Natale and L.A. Kamentsky, Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proe. Natl. Acad. Sci. U.S.A., 76 (1979) 5728-5730. I0 G.E. Kenny, Serological comparison of ten glycolytic mycoplasma species. J. Bacteriol., 98 (1969) 1044~1055. I I F. Schroeder, I Goetz and E. Roberts, Age-related alterations in cultured human fibroblast membrane structure and function. Mech. Ageing Dev., 25 (1984) 365-389. 12 S. Goldstein, E.J. Moerman and K. Porter, High-voltage electron microscopy of human diploid fibroblasts during aging/n vitro: Morphometric analysis of mitochondria. Exp. Cell. Res., 154 (1984) 101 III. 13 S. Goldstein, S.R. Ballantyne, A.L. Robson and E.J. Moerman, Energy metabolism in cultured human fibroblasts during aging in vitro. J. Cell. Physiol., 112 (1982) 112~419.