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Bilirubin increases mitochondrial inner membrane conductance
BlOCHEMKAL
MEDICINE
34, 226-229 (1985)
Bilirubin Increases Mitochondrial Inner Membrane Conductance DAVID A. STUMPF,’ Departments
of Neurology
LUIS A. EGUREN, AND JANICE K. PARKS
and Pediatrics. Sciences Center,
School of Medicine. Denver, Colorado
University 80262
of Colorudo
Health
Received September 5, 1984
Bilirubin toxicity has been ascribed to its effects on mitochondria. However, these earlier studies predate the elucidation of Mitchell’s chemiosmotic theory of oxidative phosphorylation. Mitchell proposed that the electron transport chain directs hydrogen ions out of the mitochondrial matrix, generating a transmembrane potential (A*‘) and a pH gradient (ApH). This protonmotive force (PMF) is utilized for ATP synthesis and transport processes. During the resting state (state 4). the PMF is fully developed, the electron transport chain flux minimal, and the flux of hydrogen ions across the membrane low. ADP phosphorylation utilizes the PMF, with hydrogen ions reentering the matrix through a “pore” on the ATPase. This causes a transient drop in the PMF and stimulates electron transport chain flux (state 3); the membrane hydrogen ion conductance increases. The current investigation measures bilirubin effects on the protonmotive force and membrane conductance. METHODS
Rat liver mitochondria isolation, polarographic assays, and measurements of the PMF (TPMP method) utilized our prior methods (1,2). Glutamate was the substrate used. Bilirubin (1 mM in water, adjusted to pH 7-8 with NaOH) was added followed, after 3 min, by substrates, ADP. or other reagents. In the PMF assay, mitochondria were rapidly separated from the medium by centrifugation through silicone oil (density = 1.027). This technique avoids anaerobic conditions. Calculations of polarographic and PMF results and statistical analyses (l-3) were performed on an Apple IIe computer using programs available on request. RESULTS
Bilirubin stimulated oxygen consumption in state 4 (Table 1). This increased rate was not inhibited by oligomycin but was reduced by the electron transport chain inhibitors rotenone and antimycin. There was a modest increase in the I To whom requests for reprints should be addressed: Division of Pediatric Neurology. Children‘\ Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614. 226 0006-2944/85 $3.00 Copyright All tights
Q 1985 by Academic Press. Inc. of reproduction in any form reserved.
BILIRUBIN
AND MITOCHONDRIAL
Control Bilirubin Bilirubin
(12 (24
PM) PM)
227
TABLE 1 Inner Membrane Conductance in State 4
Liver Mitochondrial Group
CONDUCTANCE
N
Oz consumption (nAtoms/min/mg protein)
PMF (mV)
17 8 8
14r 4 31? 4* 41 * 15*
209 -+ 17 216 k 9 229 2 20*
C,,H’
(nmole H’/min/mg/mV) 0.60 k 0.18 1.29 2 0.23* 1.61 k 0..54*
* Different from control. P < 0.005.
PMF at higher bilirubin concentrations (Table 2). This resulted from an increase in the pH gradient across the membrane. The inner membrane conductance dramatically increased in the presence of bilirubin, from 0.60 to 1.61 nmol/min/ mg protein/mV (Table 1). Biliverdin (12 PM) had no effect on polarographic assays. The matrix volume with 24 PM bilirubin (1.68 -t 0.91 @mg protein; mean +SD, IZ = 16) was not different from controls (1.86 2 0.73 pl/mg protein, n = 32). The yellow bilirubin color nearly all appeared in the mitochondrial pellet below the silicone oil after centrifugation; bilirubin could not be detected in the supernatant . DISCUSSION Previous investigators have reported uncoupling by bilirubin (45. This observation appeared inconsistent with the normal (6) or slightly decreased (7) ATP levels in kernicterus, although whole-brain assays in these studies might not reflect localized changes. Previous workers demonstrated abnormal coupling in brain mitochondria (8). Our polarographic assays demonstrate a similar effect in liver mitochondria. PMF measurements are required to clarify this situation. Naturally, brain mitochondria would be preferable, but a PMF assay is not available for brain mitochondria. The current studies indicate that bilirubin induces loose coupling in liver mitochondria. This state, discussed in detail elsewhere (1), is characterized by an increased inner membrane conductance but a normal PMF. A normal PMF would permit normal ATP production. This contrasts with uncoupling, in which increased oxygen consumption is associated with a reduced PMF and impaired ATP synthesis. This bilirubin effect is identical to that produced TABLE 2 Protonmotive Force in State 4 Group Control Bilirubin Bilirubin
N (12 (24
PM) PM)
17 8 8
* Different from control, P < 0.05.
PMF CmV)
A PH -0.86 -0.93 -1.11
k 0.24 ? 0.20 2 0.25*
158 ? 12 162 -I- 10 164 k 13
209 2 17 216 k 9 229 t 20”
228
STUMPF,
EGUREN,
AND PARKS
by porphyrins (1). Thus, our polarographic assay results agree with prior investigators but PMF measurements allow us to refine the interpretation. An increased oxygen consumption rate could result from the known detergent effects of bilirubin (9) and disruption of the mitochondrial membrane, However, we saw no change in matrix volume with bilirub~n, indicating an anatomically intact membrane. At higher, and probably unphysiolog~~al concentrations, b~i~bin inhibits state 3 rates as a result of effects on multiple dehydrogenases and the electron transport chain (5,lO). The relevance of in ltitro bilirubin effects to the in vivo situation has been questioned by some investigators (11,12). However, their conclusions hinge on the amount of bilirubin associated with mitochondria in viva They infused bilirubin into animals and then isolated mitochondria; little bilirubin was found in mitochondrial fractions. This is not su~r~sing because bili~bin, which is quite soluble in aqueous buffers, was probably washed out of the mitochondria. Uptake of pyrrole pigments depends on the PMF (13), which was probably compromised in these studies by use of buffers ill-suited for maintaining coupling. We did not quantitate bilirubin in our preparations, although its localization was clear. We found, by rapidly separating our coupled mitochondria from the medium, that nearly all of the yellow bilirubin color was found in the mitochondria; no yellow color was visible in the supernatant phase above the oil. Accurate assessment of the in viva dist~bution of b~i~bin will require rapid cell fractionation techniques which ensure maintenance of the PMF and minimize the loss of mitochondrial substances during isolation (14). SUMMARY Bilirubin accumulates within, and induces loose coupling in, rat liver mitochondria. This state, characterized by a normal protonmotive force, but increased oxygen consumption and inner membrane conductance, could impair cellular energy metabolism. Loose coupling is observed at bilirubin concentrations (1224 ,a~) attained in tissues of kernicteric animals. ACKNOWLEDGMENTS This work was supported by a clinical research grant from the Muscular Dystrophy Association; a grant from the National Foundation-March of Dimes; and NIH Program Project Grant HDO8315. Center Grant HDO4024, and National Research Service Award Grant HD 07096-01. all from NICHD.
REFERENCES 1. Stumpf, D. A., McCabe, E. R. B., Parks, J. K., Bullen. W. W.. and Schiff, S.. Biochenz,
Mrd,
21, 182 (1979).
2. Stumpf, D. A.. Haas, R., Eguren, L. A., Parks, J. K., and Eilert. R. E., Muscle Nerve 5. 14 (1982). 3. Stumpf, D. A., and Parks, J. K., Nertrology 29, 820 (1979). 4. Day, R. L., Proc. Sot. Exp. Biol. Med. 85, 261 (19.54). 5. Zetterstrom, R., and Emster, L., Narure (London) 178, 1335 (1956). 6. Katoh, R., Kashiwamata. S., and Niwa, F., Brain Res. 83, 81 (1975). 7. Schenker, S., McCandless, D. W., Zollman, P. E., and Wittgenstein, E., J. flin. lnvrsr. 45, 1213 (1966).
BILIRUBIN 8. 9. 10. 1I. 12. 13. 14.
AND MITOCHONDRIAL
CONDUCTANCE
229
Menken, M., Waggoner, J. G., and Berlin, N. I., J. Neurochem. 13, 1241 (1966). Emster, L., in Kemicterus (A. Sass-Knrtsak Ed.). Univ. of Toronto Press, Toronto, 1961. Karp, W. B., Pediatrics 64, 361 (1979). Brown, W. R., Grodsky, G. M., and Carbone, J. V.. Amer. J. Z’hysiol. 207, 1237 (1964). Diamond, I., and Schmid, R., Science (Washington, D.C.) 155, 1288 (1967). Keller, M.-E., Romslo, I., Biochem. J. 188, 329 (1980). Hoek, J. B., Nicholls, D. G., and Williamson, J. R.. J. Biol. Chem. 225, 1458 (1980).
MEDICINE
34, 226-229 (1985)
Bilirubin Increases Mitochondrial Inner Membrane Conductance DAVID A. STUMPF,’ Departments
of Neurology
LUIS A. EGUREN, AND JANICE K. PARKS
and Pediatrics. Sciences Center,
School of Medicine. Denver, Colorado
University 80262
of Colorudo
Health
Received September 5, 1984
Bilirubin toxicity has been ascribed to its effects on mitochondria. However, these earlier studies predate the elucidation of Mitchell’s chemiosmotic theory of oxidative phosphorylation. Mitchell proposed that the electron transport chain directs hydrogen ions out of the mitochondrial matrix, generating a transmembrane potential (A*‘) and a pH gradient (ApH). This protonmotive force (PMF) is utilized for ATP synthesis and transport processes. During the resting state (state 4). the PMF is fully developed, the electron transport chain flux minimal, and the flux of hydrogen ions across the membrane low. ADP phosphorylation utilizes the PMF, with hydrogen ions reentering the matrix through a “pore” on the ATPase. This causes a transient drop in the PMF and stimulates electron transport chain flux (state 3); the membrane hydrogen ion conductance increases. The current investigation measures bilirubin effects on the protonmotive force and membrane conductance. METHODS
Rat liver mitochondria isolation, polarographic assays, and measurements of the PMF (TPMP method) utilized our prior methods (1,2). Glutamate was the substrate used. Bilirubin (1 mM in water, adjusted to pH 7-8 with NaOH) was added followed, after 3 min, by substrates, ADP. or other reagents. In the PMF assay, mitochondria were rapidly separated from the medium by centrifugation through silicone oil (density = 1.027). This technique avoids anaerobic conditions. Calculations of polarographic and PMF results and statistical analyses (l-3) were performed on an Apple IIe computer using programs available on request. RESULTS
Bilirubin stimulated oxygen consumption in state 4 (Table 1). This increased rate was not inhibited by oligomycin but was reduced by the electron transport chain inhibitors rotenone and antimycin. There was a modest increase in the I To whom requests for reprints should be addressed: Division of Pediatric Neurology. Children‘\ Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614. 226 0006-2944/85 $3.00 Copyright All tights
Q 1985 by Academic Press. Inc. of reproduction in any form reserved.
BILIRUBIN
AND MITOCHONDRIAL
Control Bilirubin Bilirubin
(12 (24
PM) PM)
227
TABLE 1 Inner Membrane Conductance in State 4
Liver Mitochondrial Group
CONDUCTANCE
N
Oz consumption (nAtoms/min/mg protein)
PMF (mV)
17 8 8
14r 4 31? 4* 41 * 15*
209 -+ 17 216 k 9 229 2 20*
C,,H’
(nmole H’/min/mg/mV) 0.60 k 0.18 1.29 2 0.23* 1.61 k 0..54*
* Different from control. P < 0.005.
PMF at higher bilirubin concentrations (Table 2). This resulted from an increase in the pH gradient across the membrane. The inner membrane conductance dramatically increased in the presence of bilirubin, from 0.60 to 1.61 nmol/min/ mg protein/mV (Table 1). Biliverdin (12 PM) had no effect on polarographic assays. The matrix volume with 24 PM bilirubin (1.68 -t 0.91 @mg protein; mean +SD, IZ = 16) was not different from controls (1.86 2 0.73 pl/mg protein, n = 32). The yellow bilirubin color nearly all appeared in the mitochondrial pellet below the silicone oil after centrifugation; bilirubin could not be detected in the supernatant . DISCUSSION Previous investigators have reported uncoupling by bilirubin (45. This observation appeared inconsistent with the normal (6) or slightly decreased (7) ATP levels in kernicterus, although whole-brain assays in these studies might not reflect localized changes. Previous workers demonstrated abnormal coupling in brain mitochondria (8). Our polarographic assays demonstrate a similar effect in liver mitochondria. PMF measurements are required to clarify this situation. Naturally, brain mitochondria would be preferable, but a PMF assay is not available for brain mitochondria. The current studies indicate that bilirubin induces loose coupling in liver mitochondria. This state, discussed in detail elsewhere (1), is characterized by an increased inner membrane conductance but a normal PMF. A normal PMF would permit normal ATP production. This contrasts with uncoupling, in which increased oxygen consumption is associated with a reduced PMF and impaired ATP synthesis. This bilirubin effect is identical to that produced TABLE 2 Protonmotive Force in State 4 Group Control Bilirubin Bilirubin
N (12 (24
PM) PM)
17 8 8
* Different from control, P < 0.05.
PMF CmV)
A PH -0.86 -0.93 -1.11
k 0.24 ? 0.20 2 0.25*
158 ? 12 162 -I- 10 164 k 13
209 2 17 216 k 9 229 t 20”
228
STUMPF,
EGUREN,
AND PARKS
by porphyrins (1). Thus, our polarographic assay results agree with prior investigators but PMF measurements allow us to refine the interpretation. An increased oxygen consumption rate could result from the known detergent effects of bilirubin (9) and disruption of the mitochondrial membrane, However, we saw no change in matrix volume with bilirub~n, indicating an anatomically intact membrane. At higher, and probably unphysiolog~~al concentrations, b~i~bin inhibits state 3 rates as a result of effects on multiple dehydrogenases and the electron transport chain (5,lO). The relevance of in ltitro bilirubin effects to the in vivo situation has been questioned by some investigators (11,12). However, their conclusions hinge on the amount of bilirubin associated with mitochondria in viva They infused bilirubin into animals and then isolated mitochondria; little bilirubin was found in mitochondrial fractions. This is not su~r~sing because bili~bin, which is quite soluble in aqueous buffers, was probably washed out of the mitochondria. Uptake of pyrrole pigments depends on the PMF (13), which was probably compromised in these studies by use of buffers ill-suited for maintaining coupling. We did not quantitate bilirubin in our preparations, although its localization was clear. We found, by rapidly separating our coupled mitochondria from the medium, that nearly all of the yellow bilirubin color was found in the mitochondria; no yellow color was visible in the supernatant phase above the oil. Accurate assessment of the in viva dist~bution of b~i~bin will require rapid cell fractionation techniques which ensure maintenance of the PMF and minimize the loss of mitochondrial substances during isolation (14). SUMMARY Bilirubin accumulates within, and induces loose coupling in, rat liver mitochondria. This state, characterized by a normal protonmotive force, but increased oxygen consumption and inner membrane conductance, could impair cellular energy metabolism. Loose coupling is observed at bilirubin concentrations (1224 ,a~) attained in tissues of kernicteric animals. ACKNOWLEDGMENTS This work was supported by a clinical research grant from the Muscular Dystrophy Association; a grant from the National Foundation-March of Dimes; and NIH Program Project Grant HDO8315. Center Grant HDO4024, and National Research Service Award Grant HD 07096-01. all from NICHD.
REFERENCES 1. Stumpf, D. A., McCabe, E. R. B., Parks, J. K., Bullen. W. W.. and Schiff, S.. Biochenz,
Mrd,
21, 182 (1979).
2. Stumpf, D. A.. Haas, R., Eguren, L. A., Parks, J. K., and Eilert. R. E., Muscle Nerve 5. 14 (1982). 3. Stumpf, D. A., and Parks, J. K., Nertrology 29, 820 (1979). 4. Day, R. L., Proc. Sot. Exp. Biol. Med. 85, 261 (19.54). 5. Zetterstrom, R., and Emster, L., Narure (London) 178, 1335 (1956). 6. Katoh, R., Kashiwamata. S., and Niwa, F., Brain Res. 83, 81 (1975). 7. Schenker, S., McCandless, D. W., Zollman, P. E., and Wittgenstein, E., J. flin. lnvrsr. 45, 1213 (1966).
BILIRUBIN 8. 9. 10. 1I. 12. 13. 14.
AND MITOCHONDRIAL
CONDUCTANCE
229
Menken, M., Waggoner, J. G., and Berlin, N. I., J. Neurochem. 13, 1241 (1966). Emster, L., in Kemicterus (A. Sass-Knrtsak Ed.). Univ. of Toronto Press, Toronto, 1961. Karp, W. B., Pediatrics 64, 361 (1979). Brown, W. R., Grodsky, G. M., and Carbone, J. V.. Amer. J. Z’hysiol. 207, 1237 (1964). Diamond, I., and Schmid, R., Science (Washington, D.C.) 155, 1288 (1967). Keller, M.-E., Romslo, I., Biochem. J. 188, 329 (1980). Hoek, J. B., Nicholls, D. G., and Williamson, J. R.. J. Biol. Chem. 225, 1458 (1980).