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Effects of ubiquinones on spontaneous membrane hyperpolarizations in a cloned monkey kidney cell line
Life Sciences, Vol. 40, pp. 1215-1218 Printed in the U.S.A.
Pergamon Journals
EFFECTS OF UBIQUINONES ON SPONTANEOUS ~ S R A N E HYPERPOLARIZATIONS IN A CLONED MONKEY KIDNEY CELL LINE Naohide Yamashita, Hangil Chang, Kiyoshi Kurokawa and Etsuro Ogata Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo 112 Japan (Received in final form December 18, 1986)
Summary The effects of ubiquinones on spontaneous membrane hyperpolarizations in JTC-12 cells were examined. There were three types of spontaneous hyperpolarizations; rhythmic, sporadic and oscillatory types. The oscillatory type was not observed in the standard medium, whereas it was observed in sodium-free medium or in the medium containing 300 nM coenzyme Qlo" The number of the cells showing spontaneous hyperpolarizations significantly increased in the medium containing coenzyme QIO (47.7%) as compared to the control 18.9%). However, coenzyme Q1 (500 nM) showed no effects. Ubiquinone, first found in mitochondria, is an indispensable factor of the mitochondrial respiratory chain. However, ubiquinone is also present in other cell components (1,2) and thus may be important in cell function in addition to mitochondrial electron transfer (3). Ubiquinone changes the fluidity of lipid bilayers, the optimal maintenance of which is important for the regulation of physiological processes ( 3 ) . Ubiquinone stabilizes the structure of the phospholipid vesicle and protects it against phospholipase A 2 and C (4). This phenomenon may explain the protective action of ubiquinone against anoxia (4,5) . Ca ++ ions are involved in the process of anoxic injuries (41% and thus ubiquinone may play a role in the homeostasis of intracellular CaT ions. However, the effects of ubiquinone on intracellular Ca ++ ions in vitro have not yet been fully established. In the present experiment, we have examined the effects of ubiquinone on the membrane potential changes in a monkey kidney cell li~e, JTC-12, which exhibits spontaneous hy~rpolarizatio~s caused by Ca + -activated K + conducta$$e (6). Because Ca---activated K- channels are regulated by intracellular Ca ions (7), JTC-12 cel s may be a useful model to study the effects of ubiquinone on intracellular ca+l. ions. Methods JTC-12 cells were cultured at 37°C under 5% CO~ humidified air in Eagle z minimum essential medium containing 10% heat-inactlvated fetal calf serum. At subculture, the cells were seeded on 35 mm culture dishes and were subjected to electrophysiological studies after 1 to 2 days of culture. The conventional glass microelectrode technique was employed in the present experiment. The detail of the electrophysiological methods have been described elsewhere (6,8). The standard extracellular medium was composed of 128.8
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1216
Membrane Hyperpolarization by Ubiquinone
Vol. 40, No. 12, 1987
mM NaCI, 6 mM KCI, 1 mM MgCI , 2 5 mM CaCI , 5 5 mM glucose, 20 mM Hepes 2 " 2 " (Na-salt, pH=7.4) and 4 mg/ml bovine serum albumin. Two ubiquinones, coenzyme QIN and coenzyme QI, were tested. Ubiquinones, 3 mM, were dissolved in 2.2 m~yml lecithin and ~ i00 mg/ml sorbitol in distilled water and were stored. At the experiments, coenzyme Qln and Q1 were dissolved in the extracellular medium in a concentration of ~00 nM or 500 nM, respectively. In control experiments, the vehicle in the same final concentration as that of coenzyme was dissolved in the extracellular medium. All experiments were carried ~ at 30 to 32°C. Statistical analysis was done by chi-square's test. Results JTC-12 cells showed three types of spontaneous hyperpolarizations; rhythmic (Fig. IA), sporadic (Fig. 1B) and oscillatory type (Fig. IC). Spontaneous hyperpolarizations in a combination of these types were observed in some cells. The input resistance was decreased during hyperpolarizations (Fig. IA). This was the case in all types of spontaneous hyperpolar~zations. Oscillatory type of hyperpolarizations was frequently observed in+Na'-free medium in which Na ions were isoosmotically replaced with choline , whereas this type of hyperpolarizations was rarely observed in the standard medium. The effects of ubiquinones on spontaneous hyperpolarizations are shown in Table I. In the control medium, spontaneous hyperpolarization was observed in 18.9% of the cells (10/53). This value was almost identical to that in
(A)
(B)
mV
mV
--20
--20 •-40 --40
-60
(c)
-0mV -20
~
L
~
~
]-0.1nA
--40 lOsec
Fig. 1 (A) Rhythmic type of spontaneous hyperpolarization. Constant current pulse (500 msec) was applied every 3 sec. (B) Sporadic type. (C) Oscillatory type. The arrow indicates the impalement of the electrode.
Vol. 40, No. 12, 1987
Membrane Hyperpolarization by Ubiquinone
1217
TABLE I Effects of coenzyme Qlo and Q1 on spontaneous hyperpolarizations
amplitude of SH
% of cells with SH
control
18.9% (n=53)
2-14 mY
Co QI0 (300 nM)
47.7% (n=65) ~
2-40 mV
Co Q1
18.2% (n=33)
2-12 mV
(500 nM)
SH: spontaneous hyperpolarizations Co Qlo: coenzyme Q10'
Co Ql: coenzyme Q1
significant, p < 0.005
the standard medium (6), indicating that the vehicle had no effect on spontaneous hyperpolarizations. In the medium containing 300 nM coenzyme Qln, the number of the cells showing spontaneous hyperpolarizations increased t6v47.7% (31/65), which was significantly greater as compared to the control (p < 0.005). In the control medium, oscillatory type of hyperpolarizations was not observed similar to the case in the standard medium. However, in the medium containing coenzyme Q1n oscillatory type of hyperpolarizations was often recognized. In contras~Vto the effects of coenzyme QlO' coenzyme QI at the concentration up to 500 nM showed no effect on -~pontaneous i hyperpolarizations. Discussion The results in the present study showed that coenzyme Q I ~ increased the number of JTC-12 cells showing spontaneous hyperpolarizations. I n the previous study it was shown that +sp°ntane°us hyperpolarization, was caused by t~ aT permeability increase to K ions, which was medlated by intracellular C ions (6). The oscillatory type of spontaneous hyperpolarization was not observed in the control medium,+whereas it was often recognized in the medium containing coenzyme Qln" In Na -free medium, the number of the cells showing spontaneous hyperpolarlzations increased as compared to that in the standard medium (6), a response similar to the case with coenzyme QI0" In contrast with CoQ1 n, CoQ 1 showed no effect on spontaneous hyperpolarizations. It has been r e p ~ t e d t~at membrane physical changes vary with coenzyme homologs and CoQ. is incapable to restore succinate oxidation in coenzyme Q-depleted mit~chondria (9). The present result is similar to this report and thus it is considered that the effect of CoQIo is specific. In the mitochondria of hepatocytes it has been shown that there is an oscillation between NAD and NADH (i0), and that NAD stimulates the release of Ca++ ions from mitochondria while NADH inhibits it (Ii). Because coenzyme
1218
Membrane Hyperpolarization by Ubiquinone
Vol. 40, No. 12, 1987
QIO plays a role in electron-transfer chain in mitochondria, mitochondria may participate ++ in spontaneous hyperpolarizations through the regulation of intracellular Ca ions. However, it is also possible that coe~yme QI~ Iu may affect other intracellular organelle regulating intracellular Ca-- ions, such as endoplasmic reticulum. It has been reported that coenzyme Q exhibits the stabilizing effect on plasma membranes (4,5). Therefore ~ is also possible that coenzyme QI^ protected the cell from the damages of the membrane Iu occurred during the culture and at the impalement of the electrode, and this protective effect might have increased the number of the cells showing spontaneous hyperpolarizations. •
.
Acknowlegement We thank Eisai Co., Japan, for providing coenzyme QIO and QI" This work was supported by funds from Eisai Co., and a grant from the" Ministry of Education, Science and Culture of Japan. References I. 2. 3. 4. 5. 6. 7. 8.
P.S. Sastry, J. Jayaraman and T. Ramasarma, Nature (London) 189 577 (1961) J. Jayaraman and T. Ramasarma, Arch. Biochem. Biophys. 103 258-266 (1963) G. Lenaz, Coenzyme Q (G. Lenaz, ed.) pp 435-440 John Wiley & Sons Ltd., New York, (1985) T. Ozawa, Coenzyme Q (G. Lenaz, ed.) pp 441-456 John Wiely & Sons Ltd., New York, (1985) S. Marubayashi, K. Dohi, K. Ochi and T. Kawasaki, Surgery 99 184-193 (1985) H. Chang, N. Yamashita, E. Ogata and K. Kurokawa, Pflugers Archiv 405 223-225 (1985) A. Marty, Nature (London) 291 497-500 (1981) Y. Igusa, S. Miyazaki and N. Yamashita, J. Physiol. (London) 346 633-647
(1983) 9.
G. Lenaz, M.D. Esposti, R. Fato and L. Cabrini. Biomedical and Clinical Aspects of Coenzyme Q, Vol. 3 (K. Folkers and Y. Yamamura, ed.) pp 169-182 Elsevier, Amsterdam, (1981) i0. A. Boiteux and B. Hess, Farady Symp. Chem. Soc. 9 202-214 (1974) ii. A.L. Lehninger, A. Vercesi and E.A. Barabunmi, Proc. Natl. Acad. Sci. USA. 751690-1694 (1978)
Pergamon Journals
EFFECTS OF UBIQUINONES ON SPONTANEOUS ~ S R A N E HYPERPOLARIZATIONS IN A CLONED MONKEY KIDNEY CELL LINE Naohide Yamashita, Hangil Chang, Kiyoshi Kurokawa and Etsuro Ogata Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo 112 Japan (Received in final form December 18, 1986)
Summary The effects of ubiquinones on spontaneous membrane hyperpolarizations in JTC-12 cells were examined. There were three types of spontaneous hyperpolarizations; rhythmic, sporadic and oscillatory types. The oscillatory type was not observed in the standard medium, whereas it was observed in sodium-free medium or in the medium containing 300 nM coenzyme Qlo" The number of the cells showing spontaneous hyperpolarizations significantly increased in the medium containing coenzyme QIO (47.7%) as compared to the control 18.9%). However, coenzyme Q1 (500 nM) showed no effects. Ubiquinone, first found in mitochondria, is an indispensable factor of the mitochondrial respiratory chain. However, ubiquinone is also present in other cell components (1,2) and thus may be important in cell function in addition to mitochondrial electron transfer (3). Ubiquinone changes the fluidity of lipid bilayers, the optimal maintenance of which is important for the regulation of physiological processes ( 3 ) . Ubiquinone stabilizes the structure of the phospholipid vesicle and protects it against phospholipase A 2 and C (4). This phenomenon may explain the protective action of ubiquinone against anoxia (4,5) . Ca ++ ions are involved in the process of anoxic injuries (41% and thus ubiquinone may play a role in the homeostasis of intracellular CaT ions. However, the effects of ubiquinone on intracellular Ca ++ ions in vitro have not yet been fully established. In the present experiment, we have examined the effects of ubiquinone on the membrane potential changes in a monkey kidney cell li~e, JTC-12, which exhibits spontaneous hy~rpolarizatio~s caused by Ca + -activated K + conducta$$e (6). Because Ca---activated K- channels are regulated by intracellular Ca ions (7), JTC-12 cel s may be a useful model to study the effects of ubiquinone on intracellular ca+l. ions. Methods JTC-12 cells were cultured at 37°C under 5% CO~ humidified air in Eagle z minimum essential medium containing 10% heat-inactlvated fetal calf serum. At subculture, the cells were seeded on 35 mm culture dishes and were subjected to electrophysiological studies after 1 to 2 days of culture. The conventional glass microelectrode technique was employed in the present experiment. The detail of the electrophysiological methods have been described elsewhere (6,8). The standard extracellular medium was composed of 128.8
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1216
Membrane Hyperpolarization by Ubiquinone
Vol. 40, No. 12, 1987
mM NaCI, 6 mM KCI, 1 mM MgCI , 2 5 mM CaCI , 5 5 mM glucose, 20 mM Hepes 2 " 2 " (Na-salt, pH=7.4) and 4 mg/ml bovine serum albumin. Two ubiquinones, coenzyme QIN and coenzyme QI, were tested. Ubiquinones, 3 mM, were dissolved in 2.2 m~yml lecithin and ~ i00 mg/ml sorbitol in distilled water and were stored. At the experiments, coenzyme Qln and Q1 were dissolved in the extracellular medium in a concentration of ~00 nM or 500 nM, respectively. In control experiments, the vehicle in the same final concentration as that of coenzyme was dissolved in the extracellular medium. All experiments were carried ~ at 30 to 32°C. Statistical analysis was done by chi-square's test. Results JTC-12 cells showed three types of spontaneous hyperpolarizations; rhythmic (Fig. IA), sporadic (Fig. 1B) and oscillatory type (Fig. IC). Spontaneous hyperpolarizations in a combination of these types were observed in some cells. The input resistance was decreased during hyperpolarizations (Fig. IA). This was the case in all types of spontaneous hyperpolar~zations. Oscillatory type of hyperpolarizations was frequently observed in+Na'-free medium in which Na ions were isoosmotically replaced with choline , whereas this type of hyperpolarizations was rarely observed in the standard medium. The effects of ubiquinones on spontaneous hyperpolarizations are shown in Table I. In the control medium, spontaneous hyperpolarization was observed in 18.9% of the cells (10/53). This value was almost identical to that in
(A)
(B)
mV
mV
--20
--20 •-40 --40
-60
(c)
-0mV -20
~
L
~
~
]-0.1nA
--40 lOsec
Fig. 1 (A) Rhythmic type of spontaneous hyperpolarization. Constant current pulse (500 msec) was applied every 3 sec. (B) Sporadic type. (C) Oscillatory type. The arrow indicates the impalement of the electrode.
Vol. 40, No. 12, 1987
Membrane Hyperpolarization by Ubiquinone
1217
TABLE I Effects of coenzyme Qlo and Q1 on spontaneous hyperpolarizations
amplitude of SH
% of cells with SH
control
18.9% (n=53)
2-14 mY
Co QI0 (300 nM)
47.7% (n=65) ~
2-40 mV
Co Q1
18.2% (n=33)
2-12 mV
(500 nM)
SH: spontaneous hyperpolarizations Co Qlo: coenzyme Q10'
Co Ql: coenzyme Q1
significant, p < 0.005
the standard medium (6), indicating that the vehicle had no effect on spontaneous hyperpolarizations. In the medium containing 300 nM coenzyme Qln, the number of the cells showing spontaneous hyperpolarizations increased t6v47.7% (31/65), which was significantly greater as compared to the control (p < 0.005). In the control medium, oscillatory type of hyperpolarizations was not observed similar to the case in the standard medium. However, in the medium containing coenzyme Q1n oscillatory type of hyperpolarizations was often recognized. In contras~Vto the effects of coenzyme QlO' coenzyme QI at the concentration up to 500 nM showed no effect on -~pontaneous i hyperpolarizations. Discussion The results in the present study showed that coenzyme Q I ~ increased the number of JTC-12 cells showing spontaneous hyperpolarizations. I n the previous study it was shown that +sp°ntane°us hyperpolarization, was caused by t~ aT permeability increase to K ions, which was medlated by intracellular C ions (6). The oscillatory type of spontaneous hyperpolarization was not observed in the control medium,+whereas it was often recognized in the medium containing coenzyme Qln" In Na -free medium, the number of the cells showing spontaneous hyperpolarlzations increased as compared to that in the standard medium (6), a response similar to the case with coenzyme QI0" In contrast with CoQ1 n, CoQ 1 showed no effect on spontaneous hyperpolarizations. It has been r e p ~ t e d t~at membrane physical changes vary with coenzyme homologs and CoQ. is incapable to restore succinate oxidation in coenzyme Q-depleted mit~chondria (9). The present result is similar to this report and thus it is considered that the effect of CoQIo is specific. In the mitochondria of hepatocytes it has been shown that there is an oscillation between NAD and NADH (i0), and that NAD stimulates the release of Ca++ ions from mitochondria while NADH inhibits it (Ii). Because coenzyme
1218
Membrane Hyperpolarization by Ubiquinone
Vol. 40, No. 12, 1987
QIO plays a role in electron-transfer chain in mitochondria, mitochondria may participate ++ in spontaneous hyperpolarizations through the regulation of intracellular Ca ions. However, it is also possible that coe~yme QI~ Iu may affect other intracellular organelle regulating intracellular Ca-- ions, such as endoplasmic reticulum. It has been reported that coenzyme Q exhibits the stabilizing effect on plasma membranes (4,5). Therefore ~ is also possible that coenzyme QI^ protected the cell from the damages of the membrane Iu occurred during the culture and at the impalement of the electrode, and this protective effect might have increased the number of the cells showing spontaneous hyperpolarizations. •
.
Acknowlegement We thank Eisai Co., Japan, for providing coenzyme QIO and QI" This work was supported by funds from Eisai Co., and a grant from the" Ministry of Education, Science and Culture of Japan. References I. 2. 3. 4. 5. 6. 7. 8.
P.S. Sastry, J. Jayaraman and T. Ramasarma, Nature (London) 189 577 (1961) J. Jayaraman and T. Ramasarma, Arch. Biochem. Biophys. 103 258-266 (1963) G. Lenaz, Coenzyme Q (G. Lenaz, ed.) pp 435-440 John Wiley & Sons Ltd., New York, (1985) T. Ozawa, Coenzyme Q (G. Lenaz, ed.) pp 441-456 John Wiely & Sons Ltd., New York, (1985) S. Marubayashi, K. Dohi, K. Ochi and T. Kawasaki, Surgery 99 184-193 (1985) H. Chang, N. Yamashita, E. Ogata and K. Kurokawa, Pflugers Archiv 405 223-225 (1985) A. Marty, Nature (London) 291 497-500 (1981) Y. Igusa, S. Miyazaki and N. Yamashita, J. Physiol. (London) 346 633-647
(1983) 9.
G. Lenaz, M.D. Esposti, R. Fato and L. Cabrini. Biomedical and Clinical Aspects of Coenzyme Q, Vol. 3 (K. Folkers and Y. Yamamura, ed.) pp 169-182 Elsevier, Amsterdam, (1981) i0. A. Boiteux and B. Hess, Farady Symp. Chem. Soc. 9 202-214 (1974) ii. A.L. Lehninger, A. Vercesi and E.A. Barabunmi, Proc. Natl. Acad. Sci. USA. 751690-1694 (1978)